Abstract: To investigate the metabolites of a new synthetic cannabinoid 3, 3-dimethyl-2-1-(4-cyanobutyl)indazole-3-formamiminomethyl butyrate (4CN-MDMB-BUTINACA) in vitro, a human liver microsome incubation model was established to analyze the metabolic biotransformation of synthetic cannabinoids using ultra-high performance liquid chromatography coupled to quadrupole-orbitrap high-resolution mass spectrometry. Nontarget metabolomic results showed that the metabolites of 4CN-MDMB-BUTINACA included hydroxylation, ester hydrolysis, ester hydrolysis with hydroxylation reaction, pentane oxidation and ester hydrolysis with pentane oxidation reaction, among which M1-a, M2 and M4 were potential metabolic markers. The research results provide a theoretical basis and technical support for the biomonitoring and metabolic characterisation of the cannabinoid 4CN-MDMB-BUTINACA.