25B-NBOMe and 25C-NBOMe are potent 5-HT2A receptor agonists that have been associated with inducing hallucinogenic effects in drug users and severe intoxications. This paper describes the identification of their metabolites in rat and human urine by liquid chromatography (LC)-high resolution (HR)-MS/MS, the comparison of metabolite formation in vitro and in vivo and in different species, the general involvement of human cytochrome-P450 (CYP) isoenzymes on their metabolism steps, and their detectability by standard urine screening approaches (SUSAs) using GC–MS, LC–MSn, or LC-HR-MS/MS. Both NBOMe derivatives were mainly metabolized by O-demethylation, O,O-bis-demethylation, hydroxylation, and combinations as well as by glucuronidation and sulfation of the main phase I metabolites. For 25B-NBOMe, 66 metabolites could be identified and 69 for 25C-NBOMe. After application of low doses of both substances to rats, they were detectable mainly via their metabolites by both LC-based SUSAs. In case of acute intoxication, it was possible to detect 25B-NBOMe and its metabolites in an authentic human urine sample when using the GC–MS SUSA in addition to the LC-based SUSAs. Initial CYP activity screening revealed the involvement of CYP1A2 and CYP3A4 in hydroxylation and CYP2C9 and CYP2C19 in O-demethylation. The presented study demonstrated that 25B-NBOMe and 25C-NBOMe were extensively metabolized and detectable by both LC-based SUSAs.