The metabolic profiles of tryptamine-derived new psychoactive substance 5-Methoxy-N, N-diallyltryptamine (5-MeO-DALT) were investigated using both zebrafish and human liver microsome models. The ultra-high performance liquid chromatography Q Exactive Quadrupole-Orbitrap high resolution mass spectrometer (UPLC-Q-Orbitrap-HRMS) was employed to analyze the intoxicated zebrafish samples, as well as human liver microsomes samples. The mass spectrometric data were analyzed by a software of Compound Discoverer with a database of potential metabolites. As the result, A total of 11 metabolites were generated in human liver microsome model. The main metabolic pathways of the phase I metabolism included N-Oxidation,Aromatic hydroxylation,Indole-dihydroxylation reaction,N-Dealkylation and aromatic hydroxylation,N-Dealkylation and O-demethylation,Aromatic hydroxylation and O-demethylation and Hydration and N-oxidation. Meanwhile the phase II metabolism included Glucuronidation following O-demethylation,Glucuronidation following aromatic hydroxylation. A total of 8 metabolites were generated in zebrafish model. The main metabolic pathways of the phase I metabolism included Aromatic hydroxylation, N-Dehydrogenation, N-Dealkylation, N-Dealkylation and aromatic hydroxylation, meanwhile the phase II metabolism included Sulfonation following aromatic hydroxylation, Glucuronidation following aromatic hydroxylation, Sulfonation following O-demethylation. The phase I metabolites 5-MeO-DALT-Aromatic hydroxylation, 5-MeO-DALT-N-Depropylation and the phase II metabolite OH&Glucuronidation conjugation-5-MeO-DALT, OH& Sulfonation conjugation-5-MeO-DALT were proposed to be appropriate markers for 5-MeO-DAPT intake for screening, while the inclusion of the parent drug itself and OH&Glucuronidation conjugation-5-MeO-DALT may be useful for confirmation purposes.