We describe the analysis of two potent anti-human immunodeficiency virus reverse transcriptase (RT-HM inhibitors, zidovudine (AZT) and stavudine (d4T), among a pool of natural nucleosides (A, C, G, U, T) by capillary zone electrophoresis (CZE)-ionspray-tandem mass spectrometry (MS/MS) in positive ion mode. Several volatile formic acid-ammonia buffers having the same ionic strength (50 mm) but different pH values varying in the 9-11 pH range were prepared and tested to determine the best electrophoretic migration conditions. Quantitative CZE-MS/MS analysis was performed using selected reaction monitoring (SRM) mode. Finally, this CZE-MS/MS procedure opens the possibility for future determination of several nucleoside RT-HIV inhibitors in cell pool samples.