Mesaconitine is the predominant active ingredient in Aconitum carmichaelii Debx. The compound 10-hydroxy mesaconitine is one known metabolite of mesaconitine and is toxic. In order to better understand its pharmacokinetics, UPLC-MS/MS was used in this paper to measure the concentration of 10-hydroxy mesaconitine in the plasma of rats after oral (5 mg/kg) and intravenous (0.1 mg/kg) administration of 10-hydroxy mesaconitine. The concentrations of 10-hydroxy mesaconitine in rat plasma measured in the standard curve covered the range of 0.3–60 ng/mL. The intraday and interday precisions of the samples of 10-hydroxy mesaconitine in rat plasma were lower than 15%. In addition, the accuracies ranged between 96.0% and 109.3%, the matrix effects ranged between 88.9% and 98.1%, and the recoveries were all higher than 79.1%. The AUC(0 − t) values were 23.6 ± 5.9 and 207.6 ± 72.9 ng/mL·h for intravenous and oral administration, respectively, and the bioavailability of 10-hydroxy mesaconitine was 17.6%. Lastly, t1/2 was 1.3 ± 0.6 h and 3.1 ± 0.4 h for intravenous and oral administration, respectively.