Glycyrrhizae Radix (GR) is often prescribed together with Aconiti Laterlis Radix (ALR) (a so-called compatible drug pair) in traditional Chinese medicinal practice to reduce toxicity of ALR. However, the mechanisms involved remain to be addressed. In this study, the metabolic interactions between GR-ALR drug pair were investigated for the first time. First, an HPLC-TQ-MS/MS method was developed to analyze hypaconitine, a major bioactive and toxic component of ALR, in rat liver S9. Then the in vitro metabolic rates of hypaconitine by different rat liver S9 were compared using the established method. The experiments were designed in four groups: pure hypaconitine (group I) and ALR extract (group II) incubated with liver S9 of normal rats, and pure hypaconitine (group III) and ALR extract (group IV) incubated with liver S9 of GR-pretreated rats. When incubated for more than 4h, the metabolic rates of hypaconitine in group III were significantly higher than those in group I, and when incubated for more than 2h, the metabolic rates of hypaconitine in group IV were significantly higher than those in group II, suggesting that GR can enhance metabolic rate of hypaconitine, the mechanism of which might be related to hepatic metabolizing enzyme induction by GR.