The aim of our study was to demonstrate the positive impact that in vitro systems could have on the synthesis and characterization of unknown metabolites of banned doping agents. Using norandrostenedione (estr-4-en-3,17-dione), we were able to identify and characterize by GC/MS and LC/UV/MS several new hydroxylated metabolites formed in human hepatocyte incubations. The site of hydroxylation of M1, M2, M3, and M5 was demonstrated to be at C-6beta position by incubating estr-4-en-6beta-ol-3,17-dione (M4), which is the direct 6beta-hydroxylated metabolite of norandrostenedione. The structure of M5 was confirmed to be estr-4-en-6beta,17beta-diol-3-one (6beta-hydroxynortestosterone) using a commercially available authentic standard. For the other metabolites, M1, M2, and M3, no standards were available. Due to limited access to fresh human liver tissues, in vitro incubation conditions in rat liver subcellular fractions and hepatocytes were optimized as an alternative to produce sufficient quantities of the unknown metabolites for MS and/or NMR characterization. The structure of M1 was assigned to 5alpha-estran-3alpha,6beta-diol-17-one (6beta-hydroxynorandrosterone) and M3 to 5alpha-estran-3beta,6beta-diol-17-one (6beta-hydroxynorepiandrosterone) based on NMR data. M2 is proposed to be 5beta-estran-3alpha,6beta-diol-17-one (6beta-hydroxynoretiocholanolone) based on GC/MS fragmentation of the TMS-enol bis-TMS-ether derivative. The in vitro approach reported here, in addition to urinary excretion studies in humans, could contribute significantly to the discovery, the synthesis, and structure elucidation of new markers of doping agents.