Abstract3‐Methoxyeticyclidine (3‐MeO‐PCE), a phencyclidine‐type substance, has a higher N‐methyl‐D‐aspartate receptor binding affinity than phencyclidine and an involvement in fatal intoxication cases. The aim of this study was to identify new biomarkers and biotransformation pathways for 3‐MeO‐PCE. In vitro models were established using zebrafish and human liver microsomes for analysis of the phases I and II metabolites of 3‐MeO‐PCE by liquid chromatography–high‐resolution mass spectrometry. Urine samples of known 3‐MeO‐PCE consumers in forensic cases were then subjected to analysis. Overall, 14 metabolites were identified in zebrafish and human liver microsomes, allowing postulation of the following metabolic pathways: hydroxylation, O‐demethylation, N‐dealkylation, dehydrogenation, combination, and glucuronidation or sulfation. 3‐MeO‐PCE and three metabolites (M2, M3, and M6) were detected in urine. We recommended M2 (the hydroxylation product) as a potential biomarker for documenting 3‐MeO‐PCE intake in clinical and forensic cases.