AbstractGelsenicine is an indole alkaloid isolated from Gelsemium elegans Benth. In recent years, the role of G. elegans Benth preparations in anti‐tumor, analgesic, dilatation and dermatological treatment has attracted attention, and it has been applied clinically, but it is easy to cause poisoning with its use. An UPLC–MS/MS method was established to determine the gelsenicine in mouse blood, and the pharmacokinetics of gelsenicine after intravenous (0.1 mg/kg) and intragastric (0.5 and 1 mg/kg) administration was studied. Deltalin was used as internal standard; a UPLC BEH C18 column was used for chromatographic separation. The mobile phase consisted of acetonitrile and 10 mmol/L ammonium acetate (0.1% formic acid) with a gradient elution flow rate of 0.4 mL/min. Multiple reaction monitoring mode was used for quantitative analysis of gelsenicine in electrospray ionization positive interface. Proteins from mouse blood were removed by acetonitrile precipitation. A validation of this method was performed in accordance with the US Food and Drug Administration guidelines. In the concentration range of 0.05–100 ng/mL, the gelsenicine in the mouse blood was linear (r > 0.995), and the lower limit of quantification was 0.05 ng/mL. In the mouse blood, the intra‐day precision RSD was <12%, the inter‐day precision RSD was <15%, the accuracy ranged from 89.8 to 112.3%, the average recovery was >76.8%, and the matrix effect was between 103.7 and 108.4%, which meet the pharmacokinetic research requirements of gelsenicine. The UPLC–MS/MS method is sensitive, rapid and selective, and has been successfully applied to the pharmacokinetic study of gelsenicine in mice. The absolute bioavailability of gelsenicine is 1.13%.