Delsoline, a major alkaloid of Delphinium anthriscifolium Hance, has both a curare-like effect and a ganglion-blocking effect and is used to relieve muscle tension or hyperkinesia. A ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was established for the determination of delsoline in mouse blood, and the pharmacokinetics of delsoline after intravenous administration (1 mg/kg) and intragastric administration (9, 6, and 3 mg/kg) were studied. Gelsenicine served as an internal standard, and a UPLC BEH C18 chromatographic column was used. The mobile phase consisted of acetonitrile and 0.1% formic acid; the gradient elution flow rate was 0.4 mL/min. The MRM model was used for the quantitative analysis of delsoline m/z 468.3⟶108.1 and the internal standard m/z 327.1⟶296.1. Mouse blood samples were treated with acetonitrile precipitation to remove proteins. In the concentration range of 0.1–1000 ng/mL, delsoline in mouse blood showed a good linearity (r2 > 0.995), and the lower limit of quantitation was 0.1 ng/mL. The intraday precision relative standard deviation (RSD) was below 14%, and the interday precision RSD was below 15%. The accuracy ranged between 94.3% and 110.1%, the average recovery was above 90.8%, and the matrix effect ranged between 97.0% and 102.5%. The UPLC-MS/MS method was sensitive, rapid, and selective in the study of pharmacokinetics of delsoline. The absolute bioavailability of delsoline was 20.9%.