Abstract: Aristolochic acids (AAs), derived from aristolochia plant species, are a mixture of structural-related compounds. Aristolochic acid Ⅰ and aristolochic acid Ⅱ are reported to be the major components that are associated with kidney damage toxicity and carcinogenic mutagenicity toxicity. Since the toxicity mechanism of action of Aristolochic acids are still unclear, it is necessary to develop a sensitive analytical method to facilitate these studies. In this work, a rapid and sensitive method based on high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was applied for the determination of aristolochic acid Ⅰ, aristolochic acid Ⅱ and their major metabolites in samples from in vitro metabolism studies. Aristolochic acid Ⅰ and aristolochic acid Ⅱ were incubated with rat liver microsome S9 fractions under aerobic and anaerobic conditions. The incubation samples were analyzed by HPLC-MS/MS. The obtained results showed that the in vitro metabolic pathways of aristolochic acid Ⅰ and aristolochic acid Ⅱ are different. The metabolites of aristolochic acid Ⅰ was detected and characterized as aristolochic acid Ⅰa under aerobic condition and aristololactam Ⅰ under anaerobic condition, respectively; however, in the case of aristolochic acid Ⅱ, the only metabolite of aristolochic acid Ⅱ was identified as aristololactam Ⅱ under the anaerobic condition. Besides, the fragmentation behaviors of aristolochic acid Ⅰ, aristolochic acid Ⅱ and their metabolites in rat liver microsomes S9 fractions were characterized and summarized. The results indicated that the production of aristololactams is the main cause of the toxicity of aristolochic acid Ⅰ and aristolochic acid Ⅱ in the metabolism of rat liver microsome S9. This study have a certain significance for better understanding of the toxicity mechanism of action of aristolochic acid Ⅰ and aristolochic acid Ⅱ, as well as for toxicity monitoring of aristolochia plants.