(E)-Methyl-4-aryl-4-oxabut-2-enoate(YH-8) is a novel PKn B protein kinase inhibitor with good anti-tuberculosis activity. To evaluate its pharmacokinetics in rats, a sensitive and selective high performance liquid chromatography–tandem mass spectrometric(LC–MS/MS) method has been developed and validated for the quantification of YH-8 in rat plasma for the first time. Samples were pre-treated using a liquid–liquid extraction with ethyl acetate and the chromatographic separation was performed on a C18 column by gradient elution with methanol–water as the mobile phase. YH-8 was detected using a tandem mass spectrometer in positive selected reaction monitoring(SRM) mode. Method validation revealed good linearity over the range of1–500 ng/m L for YH-8 with a lower limit of quantification(LLOQ) of 1 ng/m L. Intra- and inter-day precision of YH-8 assay in rat plasma samples were 2.0%–6.8%, with accuracy of the method being 100.69%–106.18%.Stability test showed that when spiked into rat plasma, YH-8 was stable for 12 h at room temperature, for up to15 days at -70℃, and after three freeze-thaw cycles. Extracted samples were found to be stable over 12 h in an auto-sampler. The method was successfully applied to the pharmacokinetic study of YH-8 in rats after oral administration at 100 mg/kg and 200 mg/kg. (E) -Methyl-4-aryl-4-oxabut-2-enoate (YH-8) is a novel PKn B protein kinase inhibitor with good anti-tuberculosis activity. To evaluate its pharmacokinetics in rats, a sensitive and selective high performance liquid chromatography-tandem mass spectrometry (LC-MS / MS) method has been developed and validated for the quantification of YH-8 in rat plasma for the first time. Samples were pre-treated using a liquid-liquid extraction with ethyl acetate and the chromatographic separation was performed on a C18 column by gradient elution with methanol-water as the mobile phase. YH-8 was detected using a tandem mass spectrometer in positive selected reaction monitoring (SRM) mode. Method validation revealed good linearity over the range of 1- 500 ng / mL for YH-8 with a lower limit of quantification (LLOQ) of 1 ng / mL L. Intra- and inter-day precision of YH-8 assay in rat plasma samples were 2.0% -6.8% with accuracy of the method being 100.69% -106.18% .Stability test showed that when spiked i nto rat plasma, YH-8 was stable for 12 h at room temperature for up to 15 days at -70 ° C and after three freeze-thaw cycles. Extracted samples were found to be stable over 12 h in an auto-sampler. The method was successfully applied to the pharmacokinetic study of YH-8 in rats after oral administration at 100 mg / kg and 200 mg / kg.