Background: Sanwujiao pill (SWJP) is a Chinese herbal preparation widely used in China. It is an essential medicine for treating rheumatism and blood stasis. However, its safety in clinical use has always been the focus of patients because it contains toxic herbs of Aconitum carmichaelii Debx. and A. vilmorinianum Kom.Objective: To further reveal the pharmaceutical and toxic effect substances and the action mechanism of SWJPs, the metabolites and their pathways of ten Aconitum alkaloids (AAs) in the preparation at different time points after oral administration in eight organs of mice were investigated.Method: The biosamples were investigated by a four-step strategy of UPLC- Q-TOF-MS /MS technology.Results: Aconitine (AC), mesaconitine (MA), and hypaconitine (HA) were not detected in any organs. The highest concentrations of the other seven AAs occurred at 0.5 h. Yunaconitine (YAC) was not detected in the brain; all seven AAs had the lowest concentration in the brain, and the metabolism was slow in the stomach. Twelve predicted metabolites were identified, the kidney and stomach were their primary distribution locations, and the most metabolites were found at 0.5h. The main metabolic pathways of the ten AAs were demethylation, deethylation, deoxygenation, hydroxylation, and deacetylation.Conclusion: This is the first report about the metabolism of ten AAs in SWJPs in mice. Significantly, the metabolic pathways and products of four hidden toxic AAs were analyzed in vivo for the first time. The results were of great significance for the safety and effectiveness of SWJPs in clinical application.