Background: A new LC-MS/MS method for the simultaneous determination of lamivudine, zidovudine, and nevirapine in human plasma is developed using column-coupling technique. Method: Labeled compound of respective analyte was used as an internal standard. After extraction from 100 μL plasma by solid phase extraction method, analytes were separated on a C18 column coupled with a cation exchange column. Total run time was 4.5 min. A tandem mass spectrometric detection was conducted using multiple reaction monitoring under positive ionization mode with an electrospray ionization interface. The method was validated as per the FDA guidelines over the concentration range of 9.47-1466.67 ng/mL for lamivudine, 10. 32-1600.00 ng/mL for zidovudine, and 15.05-2426.67 ng/mL for nevirapine. Results: Precision was in the range 0.86-5.77 (intraday) and 1.92-8.19 (interday) while accuracy was 93.25-104.36 % (intraday) and 96.83-103.28 % (interday). Stabilities of stock in aqueous solutions and in plasma were also determined. Conclusion: The method can be applied to the pharmacokinetic study of a combination treatment.