A New method was established for simultaneous estimation of Emtricitabine, Tenofovir disoproxil fumarate and Isoniazid by RP-HPLC method. proposed analytical methods are simple, novel, economical, rapid, sensitive, reproducible and accurate for the Simultaneous estimation of Tenofovir Disoproxil Fumarate, Emtricitabine and Isoniazid in Bulk and Phamaceutical dosage form by using RP-HPLC. This Method gives reliable assay results with short analysis time using mobile phase of Acetonitrile: 0.02M Potassium Dihydrogen Orthophosphate: water (pH 5.3) in the ratio of 60: 25: 15 respectively. Retention time was found to be 2.3 min, 3.7min and 4.9 min for Tenofovir Disoproxil Fumarate, Emtricitabine and Isoniazid respectively. System suitability parameters were in the desired Limit. This method has been developed and optimized as per ICH Q2 (R1) guidelines. Keywords: Emtricitabine, Tenofovir disoproxil fumarate and Isoniazid, RP‐HPLC, Simultaneous estimation.