The aim of this study was to develop an enzyme-linked immunosorbent (ELISA) assay to quantify spiggin in the three-spined stickleback. Spiggin is a glue protein produced in the kidney of male three-spined stickleback under the control of androgens during the breeding period. Disturbances of spiggin production in male fish and abnormal induction of spiggin in female fish are considered as valuable biomarkers of exposure to (anti-)androgenic chemicals. Polyclonal antibodies against a peptide sequence of spiggin (HRD-16) were used and the specificity of the antibodies was verified by Western blotting and direct ELISA experiments. By using HRD-16 antibodies and spiggin standard preparation, a competitive ELISA was set-up and validated. This assay appears sensitive, with a detection limit of 0.5 U/mL, and specific, as shown by the competition curves, obtained by serial dilution of male and female kidney homogenates, that were parallel to the spiggin standard curves. The ability of the spiggin ELISA to quantify spiggin induction was achieved by exposing male and female three-spined sticklebacks to 0.1 and 1 microg/L of methyltestosterone. The results show a significant dose-dependent induction of spiggin in methyltestosterone-exposed female fish compared to controls.