Abstract: A method applying liquid chromatography-electrospray ionization-ion trap mass spectrometry (LC-ESI-ITMS) for the simultaneous analysis of Aconitine and its in-vitro metabolites is described. Aconitine was incubated with mouse liver microsome S₉ fraction firstly, and then the incubation sample was analyzed by LC-ESI-ITMS in full-scan mode. Aconitine and its metabolites were identified from the determination of molecular ions. The metabolites detection were confirmed from tandem mass spectrometry (MSⁿ) analysis. Comparing with the analysis of urine, 8 metabolites were identified in the mouse liver S₉ fractions by comparing with the analysis of urine of rat oral with Aconitine and referring to the literature reports.