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The chemotherapeutic drug 5 fluorouracil (5 FU) is widely used for treating solid tumors. Response to 5 FU treatment is variable with 10 30 of patients experiencing serious toxicity partly explained by reduced activity of dihydropyrimidine dehydrogenase (DPD). DPD converts endogenous uracil (U) into 56 dihydrouracil (UH(2) ) and analogously 5 FU into 5 fluoro 56 dihydrouracil (5 FUH(2) ). Combined quantification of U and UH(2) with 5 FU and 5 FUH(2) may provide a pre therapeutic assessment of DPD activity and further guide drug dosing during therapy. Here we report the development of a liquid chromatography tandem mass spectrometry assay for simultaneous quantification of U UH(2) 5 FU and 5 FUH(2) in human plasma. Samples were prepared by liquid liquid extraction with 10:1 ethyl acetate 2 propanol (v/v). The evaporated samples were reconstituted in 0.1 formic acid and 10 µL aliquots were injected into the HPLC system. Analyte separation was achieved on an Atlantis dC(18) column with a mobile phase consisting of 1.0 mm ammonium acetate 0.5 mm formic acid and 3.3 methanol. Positively ionized analytes were detected by multiple reaction monitoring. The analytical response was linear in the range 0.01 10 µm for U 0.1 10 µm for UH(2) 0.1 75 µm for 5 FU and 0.75 75 µm for 5 FUH(2) covering the expected concentration ranges in plasma. The method was validated following the FDA guidelines and applied to clinical samples obtained from ten 5 FU treated colorectal cancer patients. The present method merges the analysis of 5 FU pharmacokinetics and DPD activity into a single assay representing a valuable tool to improve the efficacy and safety of 5 FU based chemotherapy. Copyright © 2012 John Wiley Sons Ltd.