A simple, rapid and sensitive high-power liquid chromatographic (HPLC) method for analysis of 5-fluorouracil (5-FU) in patient plasma was developed and validated to study clinical pharmacokinetics (PK). Plasma sample preparation was processed with ammonium acetate buffer (pH 3.5; 0.01M) followed by liquid-liquid extraction with isopropanol/ethyl acetate (15 : 85, v/v). Extraction recovery ranged from 87.55 to 95.26%. Separation was performed using a C18 column at 25 °C with UV detection at 265 nm. The isocratic mobile phase composed of acetonitrile-ammonium acetate buffer (pH 3.5; 0.01M) (2.5 : 97.5 v/v) at a flow rate of 0.8 mL/min. Retention time was less than 7 minutes. Standard curve was linear between 0.01 - 10 μg/mL and 10 - 100 μg/mL for plasma sample. The limit of quantification was 10 ng/mL. The intra- and interday precision was below 10% (RSD). The accuracy ranged from 85.24 to 104.14%. The analysis method is rapid because it needs neither time-consuming extraction procedures nor complex chromatographic condition. The method was successfully applied to access pharmacokinetics and plasma concentration at steady state (SSC) of 5-FU. The results showed the PK and SSC of 5-FU characterized by a large interpatient variability. To increase therapeutic response and reduce toxicity, we should optimize 5-FU dose by investigating PK behavior to obtain ideal SSC..