A simple, rapid, and sensitive high performance liquid chromatographic method with UV detection has been developed and validated according to the FDA guidelines for the quantitation of zidovudine (ZDV), lamivudine (LMV), and nevirapine (NVR) in human plasma. The sample was prepared by simple liquid-liquid extraction. Chromatographic separation was carried out in a Hypersil BDS, C18 column (250 mm × 4.6 mm; 5 µm particle size) with simple mobile phase composition of 0.1 M ammonium acetate buffer in 0.5% acetic acid, v/v and methanol (40:60, v/v) at a flow rate of 0.85 ml min-1 where detector was set at 270 nm with a total run time of 10 min which is very short for simultaneous estimation of three analytes in plasma. The method was linear over the concentration range of 50–3000, 50–2000 and 10–3000 ng ml-1 with lower limit of quantifications (LLOQ) of 50, 50, and 10 ng ml-1 for ZDV, LMV, and NVR, respectively. Accuracy and precision values of both within-run and between-run obtained from six different sets of three quality control (QC) samples along with the LLOQ analyzed in separate occasions for all the analytes ranged from 94.47–99.71% and 0.298–3.507%, respectively. Extraction recovery of analytes in plasma samples was above 90.16%. In stability tests, all the analytes in human plasma were stable during storage and assay procedure. The developed and validated method was successfully applied to quantitative determination of the three analytes in plasma for pharmacokinetic study in 12 healthy human volunteers. Copyright © 2012 John Wiley & Sons, Ltd.