Abstract A sensitive and specific LC-UV method for the analysis of 5-Fluorouracil in human plasma has been developed and validated. 5-Fluorouracil was extracted from plasma by liquid-liquid method and separated on C18 reverse phase column using an elution gradient with a total run time of 11 min and detected by UV at 275 nm. For quantitative analysis of 5-Fluorouracil, 5-Bromouracil was used as internal standard. Calibration curve ranging from 9ng/mL to 2400ng/mL was obtained by enriching blank plasma pool with standard solution of 5-Fluorouracil. Within-day and between-day imprecision, as CV, ranged from 0.6% to 3.3% and from 4.4% to 13.6%, respectively. The extraction method is able to recover on average 96.8% of 5-Fluorouracil from plasma. Finally, plasma levelsof 5-Fluorouracilweredetermined in 10patients treated with 5-Fluorouracil. Results from calibration curves, quality controls and patient samples reveal that the method is suitable to analyze plasma 5-Fluorouracilfor therapeutic drug monitoring of treated patients.