Illegally added, undeclared dietary supplement ingredients represent one way of unintentional exposure to substances that are prohibited by the World Anti-Doping Agency (WADA). In addition to the risk of anti-doping rule violation, adulterated dietary supplements can also be a health risk for elite and recreational athletes. 5α-hydroxy-laxogenin is one of those illegal substances but is not prohibited yet. Though an in vitro bioassay showed androgen receptor activation, this androgenic effect could not be confirmed in the preclinical orchiectomized rat model, which might be due to a fast biotransformation into inactive metabolites. In the present study, we investigated the metabolism of 5α-hydroxy-laxogenin using three different approaches: in silico prediction using the GLORYx tool of NERRD, in vitro biotransformation using the human hepatocellular cell line HepG2 and the preclinical orchiectomized rat model, from which serum and hair samples were collected and investigated. Analyses were performed using high-performance liquid chromatography-high-resolution mass spectrometry (HPLC-HRMS). In silico metabolism predicted five different metabolites resulting from hydroxylation, reduction, or oxidation. The human HepG2 cells generated five metabolites resulting from reduction or hydroxylation and from a combination of both. Three metabolites generated in vitro and an additional mono-hydroxylated metabolite were detected in the serum of the rats treated daily subcutaneously with 5α-hydroxy-laxogenin for 2 weeks. Hair analysis points to incorporation of 5α-hydroxy-laxogenin, though showing a rather high contamination most likely by saliva or urine. In conclusion, we identified and characterized metabolites of 5α-hydroxy-laxogenin, for which, however, the exact modification sites need to be investigated in future.