Abstract A rapid, sensitive and specific method was developed and validated using liquid chromatography–tandem mass spectrometry (LC/MS/MS) for determination of gefitinib in human plasma and mouse plasma and tissue. Sample preparation involved a single protein precipitation step by the addition of 0.1 mL of plasma or a 200 mg/mL tissue homogenate diluted 1/10 in human plasma with 0.3 mL acetonitrile. Separation of the compounds of interest, including the internal standard (d8)-gefitinib, was achieved on a Waters X-Terra™ C 18 (50 mm × 2.1 mm i.d., 3.5 μm) analytical column using a mobile phase consisting of acetonitrile–water (70:30, v/v) containing 0.1% formic acid and isocratic flow at 0.15 mL/min for 3 min. The analytes were monitored by tandem mass spectrometry with electrospray positive ionization. Linear calibration curves were generated over the range of 1–1000 ng/mL for the human plasma samples and 5–1000 ng/mL for mouse plasma and tissue samples with values for the coefficient of determination of >0.99. The values for both within- and between-day precision and accuracy were well within the generally accepted criteria for analytical methods (<15%). This method was subsequently used to measure concentrations of gefitinib in mice following administration of a single dose of 150 mg/kg intraperitoneally and in cancer patients receiving an oral daily dose of 250 mg.