An expedient method is presented for determination in human plasma of gemcitabine and its metabolite 2',2'-difluorodeoxyuridine (dFdU) by ion-pair reversed-phase HPLC. Samples were simply prepared by protein precipitation. Separation was processed on a Thermo Hypersil column (250 x 4.6 mm, 5 microm Hypersil BDS C18) with UV detection at 272 nm. The mobile phase consisted of 17% methanol and 83% phosphate buffer (20 mM, pH 3.1) containing 10 mM sodium 1-heptanesulfonate with a flow rate of 0.8 mL/min. The lower limit of quantification (LLOQ) of gemcitabine was 0.08 microg/mL with linear response over the range 0.08-20.0 microg/mL, and LLOQ of dFdU was 0.1 microg/mL with linear response over the range 0.1-50.0 microg/mL. Assay accuracy for both compounds was within +/- 4%. The coefficient of variation (CV %) for intra- and interday precision for both compounds was <7%. The correlation coefficients (r2) were greater than 0.9996 for all standard curves. The simple method with adequate sensitivity has been successfully used in phase I and II gemcitabine pharmacokinetic and pharmacodynamic studies in an Asian population.