The objective of the present investigation is to develop a stability indicating UV-Spectrophotometric method for the simultaneous estimation of Emtricitabine (EMT) and Tenofovir Desoproxil Fumerate (TDF) in pure and tablet dosage forms. From the observation of the absorption spectra of EMT and TDF it was found that, TDF does not interfere with the measurement of EMT at l 1=282.4 nm, but EMT absorb appreciably along with TDF at l 2=258.7 nm. In this case the concentration of EMT is determined directly from the absorbance of the solution at 282.4 nm. The molar extinction coefficient of EMT at l 1=282.4 nm and l 2=258.7 nm is determined and found to be 9.7182x103 and 4.5672x103 lt/mol/cm and respectively. Then the absorbance contributed at 258.7 nm by this concentration of EMT is calculated from the previously known molar absorptivity of EMT at 258.7 nm, and this contribution is subtracted from the measured absorbance of the solution at 258.7, yielding the absorbance due to TDF, whose concentration is then calculated in the usual manner. The developed method was validated in terms precision, accuracy, linearity, LOD, LOQ. The system precision and method precision expressed as %RSD were found to be 1.6813&1.747 and 1.762&1.739 for EMT and TDF respectively. The mean percent of recovery in accuracy at 50,100 and 150% spiked levels were found to be 101.81, 100.00 and 100.48 for EMT and 100.74, 100.43 and 100.58 for TDF respectively. The developed method was found to be linear in the limits of 4-24 μg/ml & 6-30 μg/mL for EMT and TDF respectively. Limit of detection and limit of quantitation were found to be 0.755&2.518 and 0.332&1.108 μg/mL for EMT and TDF respectively. The developed method was applied for the determination of assay of Truvada and the percent of assay was found to be in the range of 99.75±0.291 and 99.8±0.282 for EMT and TDF respectively. A simple, rapid and economic stability indicating UV-Spectrophotometric method for simultaneous determination of EMT and TDF in bulk and formulations was developed validated. The developed method was found to be precise, accurate, linear, robust and and rugged. This method was successfully applied for the assay of Truvada; hence it can be adopted for the determination of quality in any quality control laboratory.