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UPLC/Q-TOF-MS was used for identification the metabolites of senkyunolide I in vivo.The mass errors between theoretical and experimental mass were less than 3 mDa.Eighteen metabolites in drug-treated rat plasma, urine and bile were identified.Glutathione conjugation and glucuronidation were the major pathways in vivo.Ligusticum chuanxiong Hort. (Umbelliferae) has been widely prescribed to treat cardiovascular disease in China for centuries. Senkyunolide I is one of the major bioactive components in L. chuanxiong, which shows pharmacological activities against migraines and oxidative damage. In this paper, ultra performance liquid chromatography/quadrupole-time-of-flight tandem mass spectrometry (UPLC/Q-TOF-MS) was applied for the rapid analysis of senkyunolide I metabolites in rats after its intravenous administration. The non-metabolized parent compound and eighteen metabolites from drug-treated samples in rat plasma, urine and bile were identified. Our analysis indicated that methylation, hydration, epoxidation, glucuronidation and glutathione conjugation were the major pathways of senkyunolide I metabolism in vivo. This study provides important information regarding the metabolism of senkyunolide I, which will be helpful for understanding its mechanism of action. Furthermore, this work demonstrates the potential of using UPLC/Q-TOF-MS for the rapid and reliable characterization of the metabolites of natural products.