A polyclonal antibody-based enzyme-linked immunosorbent assay (ELISA) has been developed, optimized and validated to measure this anabolic steroid. Influence of several physicochemical parameters, such as incubation time, ionic strength, detergent concentration and pH were selected to provide a highest sensitivity on the ELISA format. The regression equation of the final inhibition curve was: y = -0.3194x + 1.6316, R² = 0.9927. The linear range was between 0.1 and 25 ng/ml and the IC₅₀ was 3.5 ng/ml. The specificity was evaluated by five structurally related anabolic steroids, and none of them had significant cross-reactivity. Finally, the accuracy and precision of this assay were evaluated by means of spiked samples. The recovery was between 76.9 and 104.7%, and the variation coefficient was between 5.2 and 13.4%.