Hypaconitine (HC) is one of the main aconitum alkaloids in Aconitum carmichaelii (AC), which is considered to be effective on cardiovascular disease, although it also has high toxicity. Sini Decoction (SND), composed of Aconitum carmichaelii, Glycyrrhiza uralensis and Zingiber officinale, is a traditional Chinese multi-herbal formula for recuperating the depleted yang. The aim of this study was to compare the pharmacokinetics of HC in rat plasma after oral administration of HC, AC extract and SND, and investigate the effect of other two herbal ingredients on absorption, metabolism and elimination of HC. A sensitive and specific LC-MS/MS method was developed to determine HC in rat plasma. Eighteen male Sprague-Dawley rats were randomly assigned to three groups: HC, AC and SND group. Plasma concentrations of HC were determined at designated points after oral administration, and main pharmacokinetic parameters were estimated. It was found that there was obvious difference (p < 0.05) on the pharmacokinetic parameters among three groups. Compared with AC group, Tmax, Cmax, k, AUC(0-24) and AUC(0-∞) decreased in SND group, while t1/2 and MRT had been lengthened, which indicated that the ingredients in other two herbs could influence the pharmacokinetic behavior of HC.