To establish and optimize an in vitro incubation system with human liver microsomes and investigate the in vitro metabolites and possible metabolic pathways of mirtazapine.Three major metabolites of mirtazapine were selected to optimize the incubation conditions of liver microsomes. The metabolites of mirtazapine were analyzed by liquid chromatography-high resolution mass spectrometry (LC-HRMS) to identify the in vitro metabolites and metabolic pathways of mirtazapine.Ten metabolites, including nine phase Ⅰ metabolites and one phase Ⅱ metabolite, were identified in the in vitro liver microsome incubation. Among them, five new metabolites and one new metabolic pathway were discovered. The pathways involved in phase Ⅰ metabolic included methylation, hydroxylation, oxidation, reduction, etc., while the phase Ⅱ biotransformation was mainly glucuronidation.The metabolites discovered in this study are consistent with the main metabolites of mirtazapine reported in literature, which are N-desmethylmetazapine, 8-hydroxy mirtazapine and mirtazapine-N-oxide. The results can provide basis for the confirmation of mirtazapine cases and provide reference for the study of other substances.