Abstract: Donafenib is the deuterium derivative of sorafenib, and is an anti-tumor drug in clinical trials. An accurate and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous determination of donafenib and its N-oxide metabolite in human plasma. The analytes and internal standards (sorafenib and sorafenib N-oxide) were extracted from plasma by protein precipitation with acetonitrile, and separated on a Gemini C₁₈ (50 mm×2.0 mm, 5 μm) column using a gradient elution procedure. The mobile phase consisted of acetonitrile and 5 mmol·L^-1 ammonium acetate (0.2% formic acid) at a flow rate of 0.7 mL·min^-1. The total run time was 5.0 min. Positive electrospray ionization was performed using multiple reaction monitoring (MRM) with transitions of m/z 468.2→273.2 for donafenib and m/z 465.2→270.2 for its internal standard sorafenib, m/z 484.2→289.2 for donafenib N-oxide and m/z 481.2→286.2 for its internal standard sorafenib N-oxide. The standard curves were linear in the range of 5.00-5 000 ng·mL^-1 for donafenib, and 1.00-1 000 ng·mL^-1 for donafenib N-oxide. The method was validated and successfully applied to the pharmacokinetics study of donafenib tosylate tablets in volunteers.