Existing methods for bioanalysis of nevirapine can be improved. In this study, a high performance liquid chromatographic method for determination of nevirapine in plasma was optimized and validated. Sample preparation was done by protein precipitation using acetonitrile. The stationary phase was reverse phase C18 column (HyperClone® BDS 150 mm × 4.6 mm, 5 μm) set at 40oC. The injection volume was 30 μL. Step-gradient elution was conducted at a flow rate of 1.2 mL/minute and a run time of 11 minutes using a binary mixture of potassium dihydrogen orthophosphate (20 mM, pH 4.5) and acetonitrile. The ultraviolet detection wavelength was 270 nm. The internal standard was carbamazepine. Two calibration curves were modelled and were linear through the ranges of 0.5-2.5 μg/mL (R2=0.9978) and 2.5-25 μg/mL (R2=0.9992). The lowest limit of quantification was 0.5 μg/mL with 92% mean percentage recovery of nevirapine. The intra- and inter-day precision was 5 and 7% (coefficient of variation) respectively. The accuracy was acceptable with the relative deviation from the nominal concentrations of less than 7%. There was no interference by endogenous substances or by drugs used in HIV/AIDS patients. Nevirapine samples were stable with less than 9% loss on storage. The method met the FDA specifications. The method is simple with a short sample work up and is suitable for day to day quantification of nevirapine and in pharmacokinetics studies in resource limited settings.