Objective: A highly sensitive isocratic reverse phase high performance liquid chromatographic method was developed and validated for the estimation of Lamivudine and Tenofovir disproxil fumerate in Bulk drug and Pharmaceutical dosage forms. Method: Separation of Lamivudine and Tenofovir successfully achieved on symmetry COLUMN-ENABLE C18 G 5 μM, 250×4.6mm or equivalent utilizing HPLC Methanol and Water ph adjusted with 3.2 in the ratio of 70:30 v/v as mobile phase at a flow rate of 1mL/min and the eluates was monitored at260 nm. Result: Chromatogram showed peak at a retention time of 3.048min and 5.354min. The method was validated for system suitability, linearity, precision, accuracy, specificity, ruggedness, robustness, LOD and LOQ. Recovery of Tenofovir disproxil fumerate and Lamivudine were found to be in the range of 100.46% and 100.66% and showing linearity in the range of 10- 50 μg / ml. The S/N for LOD and LOQ for estimation of Lamivudine and Tenofovir disproxil fumerate were found to be 1.488μg / ml and 4.510 μg /ml and 3.552μg/ml and 9.763μg/ml respectively. Conclusion: Proposed method can be successfully applied for the quantitative determination of Tenofovir disproxil fumerate and Lamivudine in Bulk drug and Pharmaceutical dosage form.