A novel, accurate, specific, linear, precise and robust RP-HPLC method (stability indicating) has been developed and validated for the related substances (impurities) analysis of Dolutegravir, Lamivudine and Tenofovir Disoproxil Fumarate in tablet formulation. This research paper presents the developed method and the outcome of validation challenges. The RP-HPLC method was developed on a 250 x 4.6 mm, 5 µm, C18 column, with a gradient mode using combination of phosphate buffer and phosphoric acid in methanol and water as mobile phase, the detection was performed at 265nm and 235nm. The method was subjected to validation challenges of specificity, precision, linearity, accuracy, robustness and is demonstrated to be suitable for testing of stability samples. © 2022 Elsevier B.V., All rights reserved.