5-Fluorouracil (5-FU) has a broad spectrum of anti-tumor activity, widely applied to the treatment of can-cers. However, it is necessary to determine the plasma concentration of 5-FU in clinical practice due to its narrow therapeutic index. Therefore, a simple, economic and sensitive high-performance liquid chromatography (HPLC)method was developed and validated for the determination of 5-FU in human plasma. Ethyl acetate was chosen as ex-traction reagent. Chromatographic separation was performed on a Diamonsil C18 column (250 mm x 4.6 mm I.d., 5μm) with the mobile phase consisting of methanol and 20 mmol/L ammonium formate using a linear gradient elution at a flow rate of 0.8 mL/min. 5-FU and 5-bromouracil (5-BU) were detected by UV detector at 265 nm. The calibra-tion curve was linear over the concentration range of 5-500 ng/mL and the correlation coefficient was not less than 0.992 6 for all calibration curves. The intra- and inter-day precisions were less than 10.5% and 4.3%, respectively, and the accuracy was within ±3.7%,. The recovery at all concentration levels was 80.1±8.6%. 5-FU was stable under possi-ble conditions of storing and handling. This method is proved applicable to therapeutic drug monitoring and pharma-cokinetic studies of 5-FU in human.