A selective and rapid high-performance liquid chromatography–tandem mass spectrometry method was developed and validated for the quantification of raltegravir using raltegravir-d3 as an internal standard(IS). The analyte and IS were extracted with methylene chloride and n-hexane solvent mixture from 100 mL human plasma. The chromatographic separation was achieved on a Chromolith RP-18 e endcapped C18(100 mm 4.6 mm) column in a run time of 2.0 min. Quantitation was performed in the negative ionization mode using the transitions of m/z 443.1-316.1 for raltegravir and m/z 446.1-319.0 for IS. The linearity of the method was established in the concentration range of 2.0–6000 ng/m L.The mean extraction recovery for raltegravir and IS was 92.6% and 91.8%, respectively, and the IS-normalized matrix factors for raltegravir ranged from 0.992 to 0.999. The application of this method was demonstrated by a bioequivalence study on 18 healthy subjects. A selective and rapid high-performance liquid chromatography-tandem mass spectrometry method was developed and validated for the quantification of raltegravir using raltegravir-d3 as an internal standard (IS). The analyte and IS were extracted with methylene chloride and n-hexane solvent mixture The chromatographic separation was achieved on a Chromolith RP-18 e endcapped C18 (100 mm 4.6 mm) column in a run time of 2.0 min. Quantitation was performed in the negative ionization mode using the transitions of m / z 443.1-316.1 for raltegravir and m / z 446.1-319.0 for IS. The linearity of the method was established in the concentration range of 2.0-6000 ng / m L. The mean extraction recovery for raltegravir and IS was 92.6% and 91.8% respectively, and the IS-normalized matrix factors for raltegravir ranged from 0.992 to 0.999. The application of this method was demonstrated by a bioequivalence study on 18 healthy subjects.