A rapid, robust and sensitive HPLC method for analysis of uracil (U) and dihydrouracil (UH2) in plasma was developed using solid phase extraction and ultraviolet detection. Separation was achieved with a SymmetryShield RP18 column and an Atlantis dC18 column using a 10 mM potassium phosphate buffer as mobile phase. Compounds were eluted within 15 min without interference. Recovery was 80.4 and 80.6% for U and UH2. Calibration curves were linear from 2.5 to 80 ng/mL for U and 6.75 to 200 ng/mL for UH2. The LLQ was, respectively, 2.5 ng/mL for U, and 6.75 ng/mL for UH2. Within-run and between-run precision were less than 5.94% and inaccuracy did not exceed 7.80%. The overall procedure has been applied to correlate UH2/U ratio with dihydropyrimidine dehydrogenase activity in 165 cancer patients.