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A 200\u2009\u03bcL human plasma aliquot was spiked with a mixture of internal standards fludarabine and 5\u2010chlorouracil. A single\u2010step protein precipitation method was employed using 10% (v\/v) trichloroacetic acid in water to separate analytes from bio\u2010matrices. Volumes of 20\u2009\u03bcL of the supernatant were directly injected onto the HPLC system. Separation was achieved on a 30 \u00d7 2.1\u2009mm Hypercarb (porous graphitic carbon) column using a gradient by mixing 10\u2009m<jats:styled-content>m<\/jats:styled-content> ammonium acetate and acetonitrile\u20132\u2010propanol\u2013tetrahydrofuran (1\u2009:\u20093\u2009:\u20092.25, v\/v\/v). The detection was performed using a Finnigan TSQ Quantum Ultra equipped with the electrospray ion source operated in positive and negative mode. The assay quantifies a range from 10 to 1000\u2009ng\/mL for capecitabine, from 10 to 5000\u2009ng\/mL for 5\u2032\u2010DFCR and 5\u2032\u2010DFUR, and from 50 to 5000\u2009ng\/mL for 5\u2010FU and FUH<jats:sub>2<\/jats:sub> using a plasma sample of 200\u2009\u03bcL. Correlation coefficients (<jats:italic>r<\/jats:italic><jats:sup>2<\/jats:sup>) of the calibration curves in human plasma were better than 0.99 for all compounds. At all concentration levels, deviations of measured concentrations from nominal concentration were between \u22124.41 and 3.65% with CV values less than 12.0% for capecitabine, between \u22127.00 and 6.59% with CV values less than 13.0 for 5\u2032\u2010DFUR, between \u22123.25 and 4.11% with CV values less than 9.34% for 5\u2032\u2010DFCR, between \u22125.54 and 5.91% with CV values less than 9.69% for 5\u2010FU and between \u22124.26 and 6.86% with CV values less than 14.9% for FUH<jats:sub>2<\/jats:sub>. The described method was successfully applied for the evaluation of the pharmacokinetic profile of capecitabine and its metabolites in plasma of treated cancer patients. 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