A simple and rapid UPLC-MS/MS methodology was developed for the quantification of gefitinib and four of its metabolites in mouse plasma. The methodology showed excellent separation between gefitinib and its major metabolites. The bioanalytical assay had a limit of quantification of 15 ng/mL for both gefitinib and the O-desmethyl metabolite of gefitinib, with an upper limit of quantification of 7500 ng/mL. The assay was shown to be reliable and reproducible with the QC concentrations coefficient of variation ranging from 3.8% to 7.8% for gefitinib and 2.9% to 9.9% for the O-desmethyl metabolite. The assay was employed for quantification in plasma following IV and PO administration of gefitinib to the mouse. The derived PK data showed good correlation with the published literature.