A modified ion pair reverse-phase high-performance liquid chromatographic (HPLC) method for examining 5-fluorouracil levels in human serum was developed. The method employs the use of an internal standard and an organic extraction procedure. An isocratic mobile phase consisting of tetraethylammonium bromide, tetrabutylammonium hydrogen sulfate, methanol, sodium acetate, and phosphate buffer was used at a flow rate of 0.7 ml/min. 5-Fluorouracil was detected by UV absorption at a wavelength of 266 nm. The HPLC method described is a convenient and sensitive assay for examining 5-fluorouracil concentration in human serum.