A simple, rapid, and robust reverse phase HPLC method was developed and validated for the determination of related substances for Dolutegravir dispersible tablets 10mg. The primary goal of this research is to develop and validate a new RP-HPLC method for validating the amount of Impurity B (degradation impurity) as a related substance following USP guidelines. Dolutegravir and its impurities were separated using chromatographic conditions on a Phenyl-Hexyl (250 × 4.6 mm), 5µ column with a 45 % buffer (sodium dihydrogen phosphate dihydrate and EDTA): 49 % methanol: 6 % acetonitrile mixture and a pH of 2.5 ± 0.05 adjusted with orthophosphoric acid. The flow rate in isocratic elution mode was 1.2 mL/min. The column temperature was kept constant at 35°C, and the eluted compounds were measured at a wavelength of 258 nm using the PDA detector. According to USP guidelines, the developed method was tested and found to be stability-indicating, specific, rugged, precise, linear, accurate, and robust, with a high resolution and shorter retention time. The system suitability and other validation parameters were found to be within the limits. The method was sensitive because the LOD and LOQ demonstrate its sensitivity. The linearity curves for Dolutegravir and Impurity B were found to be linear, with a correlation coefficient of at least 0.997. The average percentage of impurities recovered ranged between 80 % to 120 %. As a result, the proposed method was found to be good and accurate for the quantitative determination of related substances associated with Dolutegravir dispersible tablets 10mg.