A sensitive, rugged and validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method is required for the reliable quantification of elvitegravir in rat pharmacokinetic samples. The objective of the current analytical work was to develop and validate LC-MS/MS method for the pharmacokinetic study after oral administration of elvitegravir in rats. The chromatographic separation was achieved isocratically using 5 mM ammonium formate (pH 3.0 ± 0.2): acetonitrile (30:70 v/v) on Phenomenex Luna 5 µ, C18 column (100 mm × 4.60 mm) with a flow rate and total run time of 0.6 mL/min and 4 min, respectively. The internal standard was elvitegravir D5. The linearity plots were analyzed by using 1/x 2 associated with a ten-point calibration standards. The range of concentration of elvtegravir for the method validation was 20.170 to 3428.942 ng/mL (r 2 ≥ 0.9976). The retention times of elvitegravir and elvitegravir D5 were 1.05 ± 0.015 min and 1.06 ± 0.015 min, respectively. The % recovery for analyte and internal standard was 80.84 ± 4.99 and 85.09 ± 3.45, respectively. The % mean stability for analyte was in the range of 97.84 to 105.31. The developed and systematically validated method was effectively implemented to the pharmacokinetic analysis of rat plasma samples following oral administration.