Background: Dolutegravir is a HIV integrase inhibitor, and multidrug and toxin extrusion transporter 1 inhibitor, and organic cation transporter 2 inhibitor. Method: A simple, rapid, accurate, robust and reproducible RP-HPLC method was developed and validated for the estimation of Dolutegravir. The quantitative analysis was carried out by using SpursilC18 (250*4.6mm, 5 μm) particle size with mobile phase composing of Phosphate buffer: Acetonitrile (70:30) ratio at a flow rate of 1.0ml/min detection wave length was carried out at 260 nm using PDA detector with injection volume 10μl, and the retention time was found to be 2.352 mins. This method was performed as per ICH guidelines and it produce linear response in the concentration ranges 20-100 ppm (R2=0.9996). The recovery studies was carried out and found to be within limits. The %RSD was found to be <2. The proposed method was statistically evaluated and can be applied for the routine analysis of Dolutegravir.