Drug discovery calls for faster method development and high-throughput analysis in supporting drug metabolism and pharmacokinetic (PK) studies, whereas the rapid, sensitive, and accurate analysis of biological samples remains a significant challenge. For analysis of complex biomatrices (e.g. plasma), liquid chromatography (LC) interfaced to mass spectrometry (MS) or tandem mass spectrometry (MS/MS) has been hampered by adverse matrix effects. For rapid assay development, it would be beneficial to improve the time-consuming method comparison and optimization steps by using generic procedures that work for a variety of compounds. However, injudiciously combining the generic procedures including protein precipitation for sample clean-up and electrospray ionization (ESI) for detection, as well as using conventional short-time isocratic or gradient LC, yield fast assay development, but often at the cost of decreased assay accuracy and increased risk of assay failure. We previously reported that the use of a mobile phase containing an extremely low concentration of ammonium formate (HCOONH4) or formic acid (HCOOH) increased analyte ESI response and controlled against matrix effects. We designated these favorable effects ‘LC-electrolyte effects’. These favorable effects can be achieved in either the positive or the negative ion ESI mode, but not for atmospheric-pressure chemical ionization (APCI). The magnitude of the LC-electrolyte effect on the analyte response depends on both the concentration of the electrolyte modifier added into the mobile phase and its identity, which is also analyte-dependent. In addition, LC is often optimized with more emphasis on improving the analytical sensitivity by concentrating the analyte on the LC column leading to a narrow and symmetric band and achieving sufficient separation between analytes and polar matrix components to avoid adverse ion suppression or enhancement of MS-MS detection. For these reasons, we proposed the so-called ‘pulse gradient system’ for conventional HPLC-based MS-MS analyses of complex biological samples, which is generic and makes method development straightforward. In order to support rapid PK studies for drug discovery, we applied the LC-electrolyte effects and the pulse gradient chromatography to the development of generic procedures that can be used to quickly generate reliable PK data for compound candidates. We herein demonstrate our approach using four model tested compounds (Compd-A,-B,-C, and-D). The analytical methods involve generic protein precipitation for sample clean-up, followed by application of fast LC gradients and the subsequent use of electrospray ionization tandem mass spectrometry (ESI-MS/MS) for individual measurement of the tested compounds in 20 μL plasma samples. Good linearity over the concentration range of 1.6 or 8-25 000 ng/mL (r2>0.99), precision (RSD, 0.45%-13.10%), and accuracy (91%-112%) were achieved through the use of a low dose of formic acid (0.4 mmol/L or 0.015‰) in the methanol/water-based LC mobile phase. The analytical method was quite sensitive, providing a lower limit of quantification of 1.6 pg on-column except for Compd-C (8 pg), and showed negligible ion suppression caused by matrix components. Finally, the assay suitability was demonstrated in simulated discovery PK studies of the tested compounds with i.v./p.o. dosing to rats. This new assay approach has been adopted with good results in our laboratory for many recent discovery PK studies. Drug discovery calls for faster method development and high-throughput analysis in supporting drug metabolism and pharmacokinetic (PK) studies, and the rapid, sensitive, and accurate analysis of biological samples remain a significant challenge. For analysis of complex biomatrices (eg plasma), liquid chromatography (LC) interfaced to mass spectrometry (MS) or tandem mass spectrometry (MS / MS) has been hampered by adverse matrix effects. For rapid assay development, it would be beneficial to improve the time-consuming method comparison and optimization steps by using generic procedures that work for a variety of compounds. However, injudiciously combining the generic procedures including protein precipitation for sample clean-up and electrospray ionization (ESI) for detection, as well as conventional short-time isocratic or gradient LC, yield fast assay development, but often at the cost of decreased assay accuracy and increased risk of assay failure. We previously reported that the use of a mobile phase containing an extremely low concentration of ammonium formate (HCOONH4) or formic acid (HCOOH) increased analyte ESI response and controlled against matrix effects. We designated these favorable effects ’LC-electrolyte effects’. be achieved in either the positive or the negative ion ESI mode, but not for atmospheric-pressure chemical ionization (APCI). The magnitude of the LC-electrolyte effect on the analyte response depends on both the concentration of the electrolyte modifier added into the mobile phase and its identity, which is also analyte-dependent. In addition, LC is often optimized with more emphasis on improving the analytical sensitivity by concentrating the analyte on the LC column leading to a narrow and symmetric band and achieving sufficient separation between analytes and polar matrix components to avoid adverse ion suppression or enhancement of MS-MS detection. For these reasons, we proposed the so-called ’ pulse gradient system ’for conventional HPLC-based MS-MS analyzes of complex biological samples, which is generic and makes method development straightforward. In order to support rapid PK studies for drug discovery, we applied the LC-electrolyte effects and the pulse gradient chromatography to develop the generic procedures that can be used to quickly generate reliable PK data for compound candidates. Welustral our approach using four model tested compounds (Compd-A, -B, -C, and-D). The analytical methods involve generic protein precipitation for sample clean-up, followed by application of fast LC gradients and the subsequent use of electrospray ionization tandem mass spectrometry (ESI-MS / MS) for individual measurement of the tested compounds in 20 μL plasma samples. Good linearity over the precision range of 1.6 or 8-25 000 ng / mL (r2> 0.99), precision (RSD 0.45% -13.10%), and accuracy (91% -112%) were achieved through the use of a low dose of formic acid (0.4 mmol / L o r 0.015 ‰) in the methanol / water-based LC mobile phase. The analytical method was quite sensitive, providing a lower limit of quantification of 1.6 pg on-column except for Compd-C (8 pg), and showed negligible ionization caused by by matrix components. Finally, the assay suitability was demonstrated in simulated discovery PK studies of the tested compounds with iv / po dosing to rats. This new assay approach has been adopted with good results in our laboratory for many recent discovery PK studies.