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Novel metabolites of the anabolic androgenic steroid 17α-methyltestosterone have been detected in HepG2 cell in vitro metabolic model and in human urine. Their detection was accomplished through targeted gas chromatography-(tandem) mass spectrometry analysis that has been based on microscale synthesized standards. The related synthesis and the gas chromatography-(tandem) mass spectrometry characterization of the analytical standards are described. All newly presented metabolites have a fully reduced steroid A-ring with either an 17,17-dimethyl-18-nor-Δ13 structure or they have been further oxidized at position 16 of the steroid backbone. Metabolites with 17,17-dimethyl-18-nor-Δ13 structure may be considered as side products of phase II metabolic sulfation of the 17β-hydroxy group of methyltestosterone or its reduced tetrahydro-methyltestosterone metabolites 17α-methyl-5β-androstane-3α,17β-diol and 17α-methyl-5α-androstane-3α,17β-diol that produce the known epimeric 17β-methyl-5β-androstane-3α,17α-diol and 17β-methyl-5α-androstane-3α,17α-diol metabolites. The prospective of these new metabolites to increase detection time windows and improve identification was investigated by applying the World Anti-doping Agency TD2021IDCR criteria. The new metabolites, presented herein, complement the current knowledge on the 17α-methyltestosterone metabolism and in some cases can be used as additional long-term markers in the frame of sport doping drug testing., (© 2023 The Authors. Drug Testing and Analysis published by John Wiley & Sons Ltd.)