A liquid chromatography–tandem mass spectrometry assay requiring a 100μL aliquot of human plasma for simultaneous determination of rilpivirine, a second generation non-nucleoside reverse transcriptase inhibitors of HIV and dolutegravir, a novel integrase stand transfer inhibitors of HIV concentrations has been developed. Sample pre-treatment is limited to protein precipitation with a mixture of methanol and zinc sulfate. After centrifugation the supernatant is injected in the chromatographic system, which consists of on-line solid phase extraction followed by separation on a phenyl-hexyl column. This 2.5min method, with its simple sample preparation provides sensitive (the limit of quantitation is 25ng/mL for each compound), accurate and precise (the intra-day and inter-day imprecision and inaccuracy are lower than 15%) quantification of the plasma concentration of these drugs and can be used for therapeutic drug monitoring in patients infected with HIV.