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To specifically quantify several metabolites of 5-fluorouracil (5-FU) and two endogenous monophosphate nucleotides, we developed an original method based on a liquid chromatography–tandem mass spectrometry (LC-MS/MS). This assay allowed the determination of: (i) the intracellular production of 5-fluoro-2′-deoxyuridine-5′-monophosphate (5-FdUMP) from 5-FU or 5-fluoro-2′-deoxyuridine (5-FdUrd), (ii) the impact of 5-FdUMP concentration on the intracellular 2′-deoxyuridine-5′-monophosphate (dUMP)/thymidine-5′-monophosphate (TMP) ratio, and (iii) the secretion extent of 5-FdUMP and 5-FU from human cultured cells by ABC transporters. Under our experimental conditions, cells were incubated with 5-FU or 5-FUrd. Then, cellular proteins were precipitated by methanol. This procedure provided high extraction recovery. In addition, to measure 5-FU and 5-FdUMP secretion from cells, we carried out quantification of these molecules in culture medium. Media were either directly injected (5-FU) or under went a solid phase extraction using Oasis Wax extraction cartridge (5-FdUMP). Separation of analytes was performed on a dC18 Atlantis 3.5 μm, (100 mm × 2.1 mm i.d) column with isocratic mode using ammonium formate buffer/methanol/water (5/5/90, v/v) as mobile phase. The run time did not exceed 6.2 min. The analytes were ionized in an electrospray interface under negative ion mode. We validated the method over a range of 2.5–150 ng mL −1 according to the compounds. Intra- and inter-assay variability was lower than 10% over seven days. All compounds were stable in cells or in culture medium when samples were stored at −20 °C for at least two weeks, and after three freeze-thaw cycles. No matrix effect was observed in both media.