A novel, simple, precise and accurate method developed for the estimation of Lamivudine and tenofovir disoproxil fumarate (TDF) in bulk drug form has been established. Lamivudine and tenofovir are well known drugs and used in treatment of HIV- Ⅰ. The method was performed by using C18 column, ODS Hypersil column with UV detection at 262nm by using Acetonitrile and water in ratio 55:45. The retention time was found to be 2.8 and 6.8 min for Lamivudine and tenofovir disoproxil fumarate (TDF). The linearity was found in range of 6- 14µg/ml for Lamivudine and 10- 50µg/ml for Tenofovir disoproxil fumarate with flow rate 1ml/min. the method was validated for linearity, accuracy, precision and robustness as per ICH guidelines. This method is suitable for simultaneous analysis for both the nucleoside analog reverse- transcriptase inhibitors Similarly, the %RSD value for precision was also found to be within the acceptable limit. The method was validated according to international conference of harmonization guidelines in terms of accuracy, precision, specificity, robustness, linearity and other aspects of analytical validation. The results of the analysis were validated statistically and recovery studies confirmed the accuracy and precision of the proposed method. Developed method was rapid and convenient which could be successfully applied for the routine control of both the component.