A new, simple, rapid, selective, precise and accurate isocratic reverse phase ultra performance liquid Chromatography assay method has been developed for estimation of Lamivudine in tablet formulations. The separation was achieved by using column Acquity UPLC BEH Phenyl (100×2.1mm, 1.7µm), in mobile phase consisted of pH 3.8 ammonium acetate buffer and methanol. The flow rate was 0.5mL.min-1 and the separated Lamivudine was detected using UV detector at the wavelength of 277 nm. The retention time of Lamivudine was noted to be 2.50 min respectively, indicative of rather shorter analysis time. The method was validated as per ICH guidelines. The proposed method was found to be accurate, reproducible, and consistent.