The present research work illustrates the development and validation of RP HPLC method for simultaneous estimation of tenofovir disproxil fumarate, emtricitabine and rilpivirine hydrochloride in bulk and formulated in a pharmaceutical dosage form as a nanosupension. Antiretroviral drug treatment is the primary line of therapy for treating HIV. The multicomponent system formulated as a nanosuspension evidenced increased hydrophilicity, potency and decreased side effects. The separation was carried out by using efficient BDS hypersil C18 HPLC column with empower software. Combination method of Precipitation—ultrasonic homogenization was used for the preparation of the nanosuspension. The mobile phase used was methanol, water, acetonitrile (80:13.4:6.6) v/v and flow rate 1mL /min. The developed method was thus validated as per ICH guidelines for various parameters whose results advocated the reliability of the method. The results for parameters viz. retention times of tenofovir disproxil fumarate, emtricitabine and rilpivirine were 3.09 min, 2.78 min and 3.68 min, linearity range was between 7.5-90, 5-60, 0.625-7.5µg/mL, respectively. Thus the new RP-HPLC method is optimum, reliable and can be used for the simultaneous estimation of tenofovir disproxil fumarate, emtricitabine and rilpivirine hydrochloride.