To study the intracellular metabolism of the prodrug 5-fluorocytosine (5FC), we developed a novel reverse-phase high-performance liquid chromatography method to simultaneously detect 5FC and its four major anabolic metabolites: 5-fluorouracil, 5-fluorouridine, 5-fluorouridine-monophosphate and 5-fluoro-2′deoxyuridine-5′-monophosphate. Separation of each compound was accomplished under isocratic conditions using a C18 column and mobile phase of formic acid-water (1 : 99 v/v). The method was validated for both accuracy and reproducibility in cell culture media. Additionally, metabolites were assessed for stability at ambient temperatures and following freeze-thaw cycles. Calibration curves were linear over a range of 1-200 μg/mL. Limit of quantification for four of the five compounds was 1 μg/mL in cell culture media (RSD < 11%). This method was successfully used to monitor intracellular conversion of 5FC to its metabolic products over a 24 h period. Copyright © 2009 John Wiley & Sons, Ltd.