A sensitive and rapid ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed to determine nintedanib in mice plasma using diazepam as the internal standard (IS). Sample preparation was accomplished through a protein precipitation procedure using acetonitrile. The analyte and IS were separated on an ACQUITY UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 μm) with the mobile phase of acetonitrile and 0.1% formic acid in water with gradient elution at a flow rate of 0.40 mL min⁻¹. The detection was performed on a triple quadrupole tandem mass spectrometer equipped with positive-ion electrospray ionization (ESI) by multiple reaction monitoring (MRM) of the transitions at m/z 540.3 → 113.0 for nintedanib and m/z 285.2 → 193.1 for IS. The linearity of this method was found to be within the concentration range of 0.1–500 ng mL⁻¹ with a lower limit of quantification of 0.1 ng mL⁻¹. Only 3.0 min was needed for an analytical run. The method described herein was superior to previous methods and was successfully applied to the pharmacokinetic study of nintedanib in mice after oral administration.