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Selective androgen receptor modulators (SARMs) represent non-steroidal agents commonly abused in human and animal (i.e. equine, canine) sports, with potential for further misuse as growth promoting agents in livestock-based farming. As a direct response to the real and possible implications of illicit application in both sport as well as food production systems, this study incorporated enzymatic hydrolysis (β-glucuronidase/arylsulfatase) into a previously established protocol while maintaining the minimal volume (200 µL) of urine sample required to detect SARMs encompassing various pharmacophores in urine from a range of species (i.e. equine, bovine, human, canine and rodent). The newly presented semi-quantitative UHPLC-MS/MS-based assay is shown to be fit-for-purpose, being rapid and offering high-throughput, with validation findings fulfilling criteria stipulated within relevant doping and food control legislation. • CCβ values determined at 1 ng mL−1 for majority of analytes. • Deconjugation step included in the method led to significantly increased relative abundance of ostarine in analysed incurred urine samples demonstrating the requirement for hydrolysis to detect a total form of emerging SARMs. • Assay amenable for use within routine testing to ensure fair play in animal and human sports and that animal-derived food is free from contamination with SARM residues.