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An UHPLC–MS/MS method for simultaneous determination of 8 tyrosine kinase inhibitors (TKIs) was developed and validated.The robust and sensitive method was successfully applied in a pharmacokinetic study in mice.The method can be expandable to the measurement of other TKIs.The purpose of this study was to develop and validate a robust and sensitive LC–MS/MS method for simultaneous determinations of various tyrosine kinase inhibitors (TKIs) in biological samples and to apply the method to their pharmacokinetic studies. Processed samples were injected into the UHPLC system coupled to an ESI-triple quadrupole mass spectrometer. The compounds were separated on an AcQuity UHPLC BEH C18 column (50 mm × 2.1 mm ID, 1.7 μm) using a gradient elution of acetonitrile/0.1% formic acid in water. The mass analysis was performed in an API 3200 Qtrap mass spectrometer via selective reaction monitoring operated under a positive scanning mode. The method was validated over a linear range of 3.13–800 nM for erlotinib; 6.25–1600 nM for sunitinib, pazopanib, and axitinib; and 12.5–3200 nM for sorafenib, dasatinib, lapatinib, and nilotinib, respectively. The intra-day and inter-day precision were <16.7% for quality control samples of the analytes at the low concentration level and <13.7% for all other concentrations. The accuracy (bias) for all analytes at three different concentration levels ranged from −12.2% to 15.0%. The recovery, matrix effect, and stability were all in the range of acceptance. Only 10 μl of blood were needed, demonstrating the method’s high sensitivity. The presented method was shown to be suitable for the analysis of serial blood samples collected from each mouse in a pharmacokinetic study, after the oral administration of 11 TKIs (each at 1 mg/kg) as a mixture.