An accurate, highly sensitive, and precise method for quantitative analysis of Dolutegravir (DOL) and Rilpivirine (RIL) by Ultra Flow High Performance Liquid Chromatography and Tandem Mass Spectroscopy in Human Plasma was proposed and validated successfully using Dolutegravir D4 as internal standard (ISTD). An aliquot of 200 ????L of plasma was mixed with internal standard dilution and extraction was performed by using Liquid – Liquid extraction Technique. Peak resolution was achieved on symmetry C18 (4.6 X 150 mm, 5µm) column. The total analytical run time was 3 minutes. Both analytes were monitored using Multiple Reaction Monitoring scan (MRM) and the mass spectrometer was operated in positive polarity mode. The method was validated for specificity, sensitivity, precession, accuracy, and other analytical parameters. The results found were satisfactory over the linear calibration range of 2.000 ng/mL to 1001.734 ng/mL. The developed method can be ready to use by scientific community for quantification of analytes in plasma samples from various clinical studies of different dose strengths.