Abstract: New psychoactive substances (NPSs) have presented health and social threats globally for more than a decade. As of November 2021, more than 1100 NPSs have been reported to the United Nations Office on Drugs and Crime Early Warning Advisory. Among these NPSs, synthetic cannabinoids are the second-largest group of substances that simulate the effects of tetrahydrocannabinol, accounting for 29% of NPS. Unlike marijuana, these synthetic cannabinoids are unpredictable and dangerous for users. The side effects include neurological, psychotic, gastrointestinal, renal, cardiovascular, and even lethal consequences. N-(1-carbamoyl-2,2-dimethylpropyl)-1-butylindazole-3-carboxamide (ADBBUTINACA) is a potent agonist of CB1 receptor agonist. It is an emerging synthetic cannabinoid that was first identified in Europe in 2019 and entered Singapore’s drug scene in January 2020. In 2021, it was one of the most common synthetic cannabinoid receptor agonists (SCRAs) seized by the Beijing Drug Control Agency. Due to the unavailable toxicological and metabolic data, there is a need to establish urinary metabolite biomarkers for detection of ADB-BUTINACA consumption and elucidate its biotransformation pathways for rationalizing its toxicological implications. In this study, the metabolisms of ADB-BUTINACA in authentic urine samples, zebrafish in vivo metabolism model and liver microsome in vitro metabolism model were analyzed by liquid chromatography-high resolution mass spectrometry. A total of 45 metabolites were tentatively identified in authentic urine samples, including 37 phase I metabolites and 8 phase II metabolites, seven of which were novel metabolites. There were 9 metabolic pathways, including amide hydrolysis (carboxamide), N-dealkylation, dehydrogenation, hydrolysis, ketone formation, hydroxylation, N-butanoic acid, dihydrodiol and glucuronidation. Hydroxylation and hydrolysis were the main metabolic pathways in authentic urine. Among these metabolites, the dihydrodiol metabolite was considered the main metabolite of ADB-BUTINACA, as it was the most abundant. By comparing the metabolites in the metabolic model and the authentic samples, it was found that the metabolites produced by the in vivo metabolic model of zebrafish were closer to the authentic samples, but the ranking of metabolites in both in vivo and in vitro metabolic models differed greatly from the authentic samples, so it was difficult to directly apply the metabolite data obtained from the in vivo and in vitro metabolic models to the analysis of the authentic samples. After ranking the peak areas of metabolites in authentic urine samples, ADB-BUTINACA prototype, dihydrodiol (M36), hydroxylation at n-butyl (M19) and hydroxylation at indazole ring (M16) were recommended as biomarkers of ADB-BUTINACA.