In this study, the use of equine liver/lung microsomes and S9 tissue fractions were used to study the metabolism of the androgenic/anabolic steroid stanozolol as an example of the potential of in vitro technologies in sports drug surveillance. In vitro incubates were analysed qualitatively alongside urine samples originating from in vivo stanozolol administrations using LC-MS on a high-resolution accurate mass Thermo Orbitrap Discovery instrument, by LC-MS/MS on an Applied Biosystems Sciex 5500 Q Trap and by GC-MS/MS on an Agilent 7000A. Using high-resolution accurate mass full scan analysis on the Orbitrap, equine liver microsome and S9 in vitro fractions were found to generate all the major phase-1 metabolites observed following in vivo administrations. Additionally, analysis of the liver microsomal incubates using a shallower HPLC gradient combined with various MS/MS functions on the 5500 Q trap allowed the identification of a number of phase 1 metabolites previously unreported in the equine or any other species. Comparison between liver and lung S9 metabolism showed that the liver was the major site of metabolic activity in the equine. Furthermore, using chemical enzyme inhibitors that are known to be selective for particular isoforms in other species suggested that an enzyme related to CYP2C8 may be responsible the production of 16-hydroxy-stanozolol metabolites in the equine. In summary, the in vitro and in vivo phase 1 metabolism results reported herein compare well and demonstrate the potential of in vitro studies to compliment the existing in vivo paradigm and to benefit animal welfare through a reduction and refinement of animal experimentation.