A capillary electrophoresis-ion trap mass spectrometry method with a time-segment program was developed to simultaneously analyze Ziagen and its phosphorylated metabolites such as carbovir monophosphate, carbovir diphosphate, and carbovir triphosphate. By using the time-segment program, the positively charged nucleoside analog and negatively charged nucleotides were separated and detected in a single electrophoretic run. The limits of detection were less than 2 micro M for all of the analytes. Calibration curves of the compounds showed excellent linearity over the range of 2-100 micro M. The capability of the method was demonstrated by analyzing Ziagen and its phosphorylated metabolites that were spiked in cellular extracts of human peripheral blood mononuclear cells at 20 micro M levels. Some endogenous nucleotides such as adenosine 5'-triphosphate, adenosine 5'-diphosphate, and adenosine 5'-monophosphate, were also detected in the cellular extracts.