Nucleoside reverse transcriptase inhibitors (NRTIs) have been the first class of antiretroviral drugs used against HIV infection. Despite NRTI-free regimens have been eagerly sought over the years in an effort for treatment simplification, NRTIs remain in most antiretroviral combination treatment. There has been generally a limited interest for their therapeutic drug monitoring, arguably because NRTIs levels measured in plasma poorly predict the concentration of pharmacologically active metabolites in cells. Plasma concentrations do impact cellular levels, while large differences between NRTIs have been found with regard to their ability to distribute into the cerebrospinal fluid (CSF) compartment. The renewed interest for the measurements of NRTIs concentrations in plasma and CSF was raised by ongoing efforts to understand some instances of toxicity or for determining their actual implication in the development of HIV-associated neurological disorders. In this context, a 5-min multiplex ultra-high-pressure chromatography tandem mass spectrometry (UHPLC-MS/MS) analysis in human plasma and CSF was developed for NRTIs used in clinical practice: abacavir, emtricitabine, lamivudine, tenofovir and zidovudine along with zidovudine glucuronide (Gln-ZDV). The 200-fold dilution of blank human plasma was shown to be a reliable surrogate matrix for quantification of NRTIs and Gln-ZDV in CSF. Both methodologies were fully validated over the clinically relevant concentrations, and satisfactorily fulfilled all parameters for bioanalytical methods validation. This sensitive, rapid, and robust UHPLC-MS/MS assay offers a methodology for increasing our understanding of the ability of NRTIs to cross the blood-brain barrier and their potential implication in neuropsychological disorders observed in HIV-infected patients.