A robust new analytical method has been developed for the determination of 5-fluorouracil (5-FU) in human plasma samples using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The method is based on a liquid-liquid extraction procedure, precolumn derivatization, reversed-phase HPLC separation, and detection using atmospheric pressure chemical ionization and selected reaction monitoring. The derivatization agent used was 4-bromomethyl-7-methoxycoumarin. The internal standard for the assay procedure was a stable isotope labeled analog of 5-FU. The lower limit of quantitation was 1. 0 ng/mL using 500 µ L aliquots of plasma. Sample throughput on the mass spectrometer was approximately 17 samples/h (3. 5 min/sample). The method was fully validated. The recovery of 5-FU averaged 76. 1%. The accuracy of the assay, assessed from quality control samples, ranged from 99. 1% to 104. 3% (% theoretical). The overall interassay precision (% RSD) was 2. 7%, and the intraassay precision (% RSD) ranged from 1.5% to 3. 9%. The derivatized samples were found to be stable under sample analysis conditions and during refrigerator storage. The method was specific for the determination of 5-FU.