Purpose Norflurazepam, also a metabolite of other prescription benzodiazepines, appeared on the new psychoactive substances (NPS) drug market recently, complicating the interpretation of NPS findings. The aims of the study were to tentatively identify potential metabolites of norflurazepam and structural analogues (flurazepam, fludiazepam and cinolazepam) produced by hepatocytes and in authentic human samples and to discuss the possibility to differentiate drug consumption. Methods Each drug (5 mu mol/L) was incubated with pooled human hepatocytes, and metabolites were identified using liquid chromatography-high-resolution mass spectrometry (LC-HRMS). Similarly, urine with/without hydrolysis and blood/serum from three flurazepam and seven presumptive norflurazepam cases were analysed by LC-HRMS. Results No metabolites were detected for norflurazepam in hepatocytes, but six metabolites for flurazepam, two for fludiazepam and three for cinolazepam were found. In human specimens collected after flurazepam ingestion, a total of eight metabolites, in good agreement with hepatocyte metabolites, were detected. In specimens of presumptive norflurazepam intake, norflurazepam and its metabolites (four hydroxy metabolites and one glucuronide of a hydroxy metabolite) were found. Conclusions Based on the results, hydroxy metabolites for norflurazepam, N-(hydroxyethyl), desethyl and didesethyl for flurazepam, hydroxy for fludiazepam and glucuronides and N-(hydroxyethyl) for cinolazepam are recommended for monitoring. While flurazepam, fludiazepam and cinolazepam were metabolised by hepatocytes at side chain, norflurazepam was not, which seems to indicate that hepatocytes have difficulty in modifying the benzene/diazepine rings of some 1,4-benzodiazepines. As for confirming the intake of norflurazepam, the urine ratio of 3-hydroxy-norflurazepam/norflurazepam might be the key; a high ratio might be correlated to norflurazepam intake, thereby enabling the differentiation.