Abstract: Ixerin Z and 11,13α-dihydroixerin Z are the important ingredients of sesquiterpene lactones in Kudiezi. In this study, the metabolites of Ixerin Z and 11,13α-dihydroixerin Z in rat liver microsomes were screened and identified using high performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometry (UHPLC-LTQ/Orbitrap MS) at negative ion mode. The separation was conducted on Waters Acquity UPLC BEH C18 column (2.1 mm×50 mm×1.7 μm) with the gradient elution of 0.1% formic acid aqueous solution (A) and acetonitrile (B). As a result, Ixerin Z, 11,13α-dihydroixerin Z and 16 metabolites were selected out, among which 15 metabolites were characterized based on the retention time, accurate mass measurements and fragmentation patterns. The results showed that there were two possible metabolic pathways of Ixerin Z in rat liver microsomes: 1) Metabolites 2, 5 and 12 were generated from Ixerin Z after hydroxylation; 11,13α-dihydroixerin Z was generated from Ixerin Z after hydrogenated on its double bond, then metabolites 8, 14 and 15 were generated from 11,13α-dihydroixerin Z after hydroxylation. 2) The metabolites were generated from the prototype via addition reaction, such as cysteine combination (1 and 4), glutathione combination (7 and 9). There were also two possible metabolic pathways of 11,13α-dihydroixerin Z in rat liver microsomes: 1) Ixerin Z was generated from 11,13α-dihydroixerin Z after hydrogenated on its double bond, then the metabolites 12 was generated from Ixerin Z after hydroxylation. Metabolites 3, 8, 10, 13, 14 and 15 were generated from 11,13α-dihydroixerin Z after hydroxylation, followed by hydrogenation to produce 6 and 11. 2) Metabolite 7 was generated in conjugation reaction. The results demonstrate that they mainly undergo oxidation, reduction, hydrolysis via phase I in rat liver microsomes, which will help to clarify the metabolic pattern of Ixerin Z and 11,13α-dihydroixerin Z, and can also provide support for further new drug and toxicology research.