A simple, precise, accurate stability-indicating Ultra-Performance Liquid Chromatography (UPLC) method was developed and validated for the simultaneous determination of known potential impurities present in Stavudine (ST), Lamivudine (LA) and Nevirapine (NE) in fixed dose combination drug. The desired chromatographic separation was performed on Acquity UPLC HSS-T3 (2.1 × 100 mm), 1.8 μ column using gradient elution of 0.1% perchloric acid in water adjusted the pH 2.6 with diluted sodium hydroxide solution as mobile phase A and acetonitrile as mobile phase B at flow rate of 0.2 ml/min. UV detection was performed at 265 nm. Total run time was 18 min in which main actives and eleven known and major unknown impurities were separated. The method was validated according to the International Conference on Harmonization (ICH) guidelines with respect to specificity, precision, accuracy, linearity, robustness, Limit of Detection (LOD) and Limit of Quantification (LOQ). The developed and validated Reverse Phase-Ultra Performance Liquid Chromatography (RP-UPLC) method is specific for quantification of all potential impurities present in the stavudine/lamivudine/nevirapine tablets.