This study investigates the human metabolism of four synthetic benzimidazole opioids: isotonitazene, metonitazene, etodesnitazene, and metodesnitazene. The research aims to identify specific metabolite biomarkers of consumption to document use in clinical and forensic settings. The study used 10-donor-pooled human hepatocytes to incubate the drugs and analyzed the metabolites using liquid chromatography-high-resolution tandem mass spectrometry (LC-HRMS/MS). Twelve, nine, twenty-two, and ten metabolites were identified for isotonitazene, metonitazene, etodesnitazene, and metodesnitazene, respectively. The main transformations included N-deethylation, O-dealkylation, and further O-glucuronidation. In vitro and autopsy results were consistent, validating the human hepatocyte model for predicting human metabolism. The study suggests that the parent drug and its corresponding O-dealkyl- and N-deethyl-O-dealkyl metabolites are biomarkers of exposure in urine after glucuronide hydrolysis, while the N-deethyl metabolite is an additional biomarker in blood. The findings highlight the importance of identifying specific metabolite biomarkers to better document clinical and forensic cases involving these synthetic opioids.This study investigates the human metabolism of four synthetic benzimidazole opioids: isotonitazene, metonitazene, etodesnitazene, and metodesnitazene. The research aims to identify specific metabolite biomarkers of consumption to document use in clinical and forensic settings. The study used 10-donor-pooled human hepatocytes to incubate the drugs and analyzed the metabolites using liquid chromatography-high-resolution tandem mass spectrometry (LC-HRMS/MS). Twelve, nine, twenty-two, and ten metabolites were identified for isotonitazene, metonitazene, etodesnitazene, and metodesnitazene, respectively. The main transformations included N-deethylation, O-dealkylation, and further O-glucuronidation. In vitro and autopsy results were consistent, validating the human hepatocyte model for predicting human metabolism. The study suggests that the parent drug and its corresponding O-dealkyl- and N-deethyl-O-dealkyl metabolites are biomarkers of exposure in urine after glucuronide hydrolysis, while the N-deethyl metabolite is an additional biomarker in blood. The findings highlight the importance of identifying specific metabolite biomarkers to better document clinical and forensic cases involving these synthetic opioids.