A new oral drug containing an unknown anabolic androgenic steroid (AAS) was studied. The principal constituent of the unknown anabolic hormone was studied by infrared spectrum (IR), nuclear magnetic resonance spectroscopy (1H NMR), ultraviolet spectroscopy (UV), and mass spectroscopy (MS). It was inferred to be methyl-1-testosterone (M1T, 17beta-hydroxy-17alpha-methyl-5alpha-androst-1-en-3-one) which was just added to the prohibited list in 2006. In addition, a monitoring, screening and confirmation of methyl-1-testosterone was established. The detection limit (S/N = 3) was 2 ng/mL, and the limit of quantification (S/N = 10) was 10 ng/mL. The relative standard deviation was 9.8% (n = 7) for the determination of pretreated urine sample with internal standard. This method was successfully applied in the identification of M1T positive urine. The excretion curve of M1T in human urine is described. It is a significant work for the discovery, determination and monitoring of the new AAS.