A selective, sensitive, rugged, and high throughput liquid chromatography tandem mass spectrometry method is developed for the determination of one nucleotide tenofovir (TFV) and two nucleosides emtricitabine (FTC) and lamivudine (3TC) reverse transcriptase inhibitors in human plasma. Plasma samples were prepared by solid-phase extraction of the analytes and acyclovir (ACV) as internal standard using Waters Oasis MCX cartridges. The chromatographic separation is achieved in a run-time of 3.0 min on an ACE 5 CN column (150 mm × 4.6 mm, 5 μm) under isocratic conditions. The mobile phase consisted of 0.5% formic acid in water and acetonitrile (55:45, v/v). The protonated precursor --> product ion transitions for TFV, FTC, 3TC, and internal standard were monitored on a triple quadrupole mass spectrometer operating in the multiple reaction monitoring (MRM) and positive ion mode. A linear dynamic range of 4.0-802 ng/mL, 15.0-3006 ng/mL, and 20.1-4023 ng/mL is established for TFV, FTC, and 3TC, respectively, using 0.2 mL plasma sample. The method is fully validated for its sensitivity, selectivity, accuracy and precision, ion suppression, matrix effect, recovery, stability, and dilution integrity. It is successfully applied to a bioequivalence study of [300(TFV) + 200(FTC) + 300(3TC)] mg tablet formulation in 43 healthy human subjects under fasting conditions.