A rapid, specific and high sensitive liquid chromatography tandem mass spectrometry (LCMS/MS) method was developed for the simultaneous determination of Abacavir and Lamivudine in K3EDTA human plasma. The method involves simple, solid phase extraction procedure and separation with a C18 column (5 µm, 150 × 4.6 mm) with Acetonitrile/10mM Ammonium Formate [80/20, V/V] isocratic elution at a flow-rate of 1.0 mL/min with a total run time of 3.0 minutes. Labeled isotopes were used as the internal standard. The protonate of analyte’s were quantitated in positive ionization by multiple reaction monitoring with a mass spectrometer. The mass transitions 287.200/191.100 (m/z) and 230.100/112.100 (m/z) were used to measure Abacavir and Lamivudine, respectively. The method was developed and validated using 100 µL of plasma, over a concentration range of 5.005 – 4004.253 ng/mL for Abacavir and 2.506-2005.148 ng/mL for Lamivudine. The intra and inter day precision and accuracy values were found to be within the assay variability limits as per regulatory guidelines. The developed assay method was applied to a clinical pharmacokinetic study in human volunteers.