Due to the potential for misuse of a wide range of anabolic steroids in horse racing, a screening test to detect multiple compounds, via a common class of metabolites, would be a valuable forensic tool. An enzyme-linked immunosorbent assay (ELISA) has been developed to detect 17α-alkyl anabolic steroid metabolites in equine urine. 16β-Hydroxymestanolone (16β,17β-dihydroxy-17α-methyl-5α-androstan-3-one) was synthesised in six steps from commercially available epiandrosterone (3β-hydroxy-5α-androstan-17-one). Polyclonal antibodies were raised in sheep, employing mestanolone (17β-hydroxy-17α-methyl-5α- androstan-3-one) or 16β-hydroxymestanolone conjugated to human serum albumin, via a 3-carboxymethyloxime linker, as antigens. Antibody cross-reactivities were determined by assessing the ability of a library of 54 representative steroids to competitively bind the antibodies. Antibodies raised against 16β-hydroxymestanolone showed excellent cross-reactivities for all of the 16β,17β-dihydroxy-17α-methyl steroids analysed and an ELISA has been developed to detect these steroid metabolites. Using this 16β-hydroxymestanolone assay, urine samples from horses administered with stanozolol (17α-methyl-pyrazolo[4′,3′:2,3]-5α-androstan- 17β-ol), were analysed raw, following β-glucuronidase hydrolysis, and following solid-phase extraction (SPE) procedures. The suppressed absorbances observed were consistent with detection of the metabolite 16β- hydroxystanozolol. Positive screening results were confirmed by comparison with standard LCMS analyses. Antibodies raised against mestanolone were also used to develop an ELISA and this was used to detect metabolites retaining the parent D-ring structure following methandriol (17α-methylandrost-5-ene-3β, 17β-diol) administration. The ELISA methods developed have application as primary screening tools for detection of new and known anabolic steroid metabolites.