A simple, rapid, sensitive and novel technique for quantifying of Dolutegravir in human plasma was developed and validated. A protein precipitation technique was used as sample preparation method and Tenofovir is employed as Internal standard (IS). Chromatographic condition was conducted using a Agilent, Zorbax, XDB C18 2.1 × 50 mm, 5 μm., 5 Micro meter, and a Mobile Phase (70: 30) of proportion Acetonitrile;1% Formic acid delivered at 1.20 ml/min and injection volume of 10 μL with run time of 3 minutes. ESI-MS with positive ion (PI) mode at atmospheric pressure was employed to carry out detection. The validated Linearity range is 2ng to 1000ng/ml with correlation value of 0.998 , excellent accuracy and precision was obtained. The retention time of Dolutegravir and Tenofovir 1.37 and 1.20 min.