In this study, metenolone metabolic profiles were investigated. Metenolone was administered to one healthy male volunteer. Liquid–liquid extraction and direct-injection were applied to processing urine samples. Urinary extracts were analyzed by liquid chromatography quadrupole time-of-flight mass spectrometry (LC–QTOFMS) using full scan and product ion scan with accurate mass measurement for the first time. Due to the lack of useful fragment ion for structural elucidation, GC–MS instrumentation was employed to obtain structural details of the trimethylsilylated phase I metabolite released after hydrolysis, and the EI mass spectrum was always informative in steroidal structure studies owing to more useful fragment ions than the ESI mass spectrum. 16 metabolites including 6 glucuronide and 9 unreported sulfate conjugates were characterized and tentatively identified. All the metabolites were evaluated in terms of how long they could be detected. The sulfate conjugate S6 (1-methylen-5α-androst-3,17-dione-2ξ-sulfate) was considered to be a new long term metabolite for metenolone misuse that could be detected 40 days by liquid–liquid extraction and up to 30 days by direct-injection analysis after oral administration.