Abstract: OBJECTIVE To develop a ultra high-performance liquid chromatography-tandem mass spectrometry separation method(UPLC-MS/MS) for the pharmacokinetic study of Axitinib after intravenous administration. METHODS Plasma samples were treated by acetonitrile precipitation. The effective UPLC-MS/MS separation of the examined compounds was applied on an Acquity BEH C₁₈ column (2.1 mm×50 mm, 1.7 μm) column with a gradient mobile phase system consisting of 0.1% formic acid in water and acetonitrile. The analysis was performed with a flow rate of 0.3 mL·min^-1. An electrospray ionization (ESI) was used to detect in a positive ion mode. The scanning mode was MRM. RESULTS The assay was validated over concentration ranges of 0.1-100 ng·mL^-1, with a lower limit of quantification (LLOQ) of 0.1 ng·mL^-1. Intra-and inter-assay precision values for replicate quality control samples were within 5.56%. Assay recoveries of Axitinib was higher than 87.87%. Axitinib were stable in rat plasma for at least 24 h at room temperature, 30 days at 4℃ and -20℃, and following at least three freeze-thaw cycles. Furthermore, no notable matrix effect was observed for Axitinib. CONCLUSION The accurate and simple method developed can be provided the basis and applied to clinical pharmacokinetic study.