A simple reliable analytical method was developed to determine dolutegravir sodium, lamivudine and tenofovir disoproxil fumarate in pharmaceutical effluents which are releasing into domestic water bodies by using reverse phase High Performance Liquid Chromatography (HPLC) with ultraviolet absorption detection. The proposed method was quite reproducible and sensitive enough to detect the compounds at less than 10 ppm level, which can replace the troublesome non-reproducible conventional analytical methods like UV-Visible spectrophotometric analysis or titrimetric analysis. A Reversed-Phase HPLC method was developed and validated for the estimation of dolutegravir sodium, lamivudine and tenofovir disoproxil fumarate in effluents or pharmaceutical industry washouts. The separation was achieved on C18 symmetry C-18 column (250 × 4.6 mm i.d., 5.0 μm) using sodium dihydrogen phosphate with SDS as ion pair reagent having a pH of 2.0 as mobile phase A and acetonitrile and methanol as mobile phase B in gradient mode as mobile phase and at a flow rate of 1.0 ml/min. Detection was carried out using a UV detector at 260 nm. The total chromatographic analysis time per sample was about 30.0 min with dolutegravir sodium, lamivudine and tenofovir disoproxil fumarate eluting at retention time of about 5.2 min for lamivudine, 11.0 min for dolutegravir sodium and 13.0 min for tenofovir disoproxil fumarate. The method was validated for accuracy, precision, specificity, linearity and sensitivity. Validation studies demonstrated that this HPLC method is accurate, specific, rapid, reliable and reproducible. Linearity was observed for dolutegravir sodium, lamivudine, and tenofovir disoproxil fumarate in the concentration range of 0.05-7.5 μg/ml (R2 > 0.95), the Limit of Detection (LOD) and Limit of Quantitation (LOQ) was found to be for 0.017 μg/ml and 0.053 μg/ml respectively for dolutegravir sodium, 0.016 μg/ml and 0.048 μg/ml for lamivudine and 0.018 μg/ml and 0.054 μg/ml for tenofovir disoproxil fumarate, the method was validated as per ICH guidelines. The RSD for intra-day and inter-day precision were found to be less than 5%. The percentage recovery was in good agreement and the method is simple, specific, precise, and accurate for the determination of dolutegravir sodium, lamivudine and tenofovir disoproxil fumarate in the pharmaceutical industry washouts.