Abstract: To investigate the metabolic profiles of 4′-chlorodiazepam and diclazepam in vivo, the liver microsomal metabolism model was established. Firstly, the target substances were incubated in the water bath pot at 37 ℃ for about 4 h. Then the incubation buffer was terminated by ice acetonitrile. After that 10 μL of the supernatant was taken for analysing. The Agilent Eclipse plus C18 column (12.5 mm×2.1 mm×5 μm) was used and the 20 mmol/L ammonium acetate-0.1% formic acid-5% acetonitrile (A) and acetonitrile (B) were served as the gradient elution. The metabolites and metabolic pathways were identified and clarified by liquid chromatography coupled with quadrupole Exactive Orbitrap mass spectrometry (LC-Q-Exactive MS) in the full scan-ddMS2 ion mode. The results showed that 4′-chlorodiazepam produced 5 phase Ⅰ and 2 phase Ⅱ metabolites in Wistar liver microsomes, and diclazepam produced 6 phase Ⅰ and 1 phase Ⅱ metabolites in Wistar liver microsomes. The differences of the metabolites might result from the substitution position of chlorine atom. And the main metabolic pathways of 4′-chlorodiazepam and diclazepam included demethylation, mono-hydroxylation, di-hydroxylation, O-glucuronidation in this work. On the basis of experimental results, phase Ⅰ metabolites M2 (demethylation), M3 (mono-hydroxylation), M4 (demethylation and mono-hydroxylation) of 4′-chlorodiazepam and M2′ (demethylation), M3′ (mono-hydroxylation), M4′ (demethylation and mono-hydroxylation) of diclazepam were proposed to be potential poisoning markers due to the high concentration level. Moreover, the metabolites and metabolic pathways of di-hydroxylation were the first reported. This study preliminarily clarified the metabolic pathways and main metabolites of 4′-chlorodiazepam and diclazepam in Wistar liver microsomes. It provided a theoretical basis for the pharmacokinetic and forensic scientific research of 4′-chlorodiazepam and diclazepam, which offered great value for the detection of related cases in laboratory.