Abstract: This study was designed to investigate the metabolites of 5F-AMB by human liver microsomes model in vitro. 5F-AMB was added in the reaction mixture to simulate the metabolic process in human hepatocytes in vivo, and then to determine the reaction points and pathways of metabolism by ultra performance liquid chromatography (UPLC) coupled to high resolution mass spectrum (HR-MS). 5F-AMB generated 9 metabolites in total in the human liver microsomes model. Ester hydrolysis, combination of ester hydrolysis and oxidative defluorination, combination of ester hydrolysis and hydroxylation on pentyl chain moiety and combination of ester hydrolysis and hydroxylation on indazole ring moiety reactions were its main metabolic pathways. The method is fast and efficient so that the ester hydrolysis, combination of ester hydrolysis and oxidative defluorination, combination of ester hydrolysis and hydroxylation on pentyl chain moiety and combination of ester hydrolysis and hydroxylation on indazole ring moiety metabolites of 5F-AMB can be used as the suitable and potential biomarkers in the urine samples.