A method for the analysis of testosterone, androstenedione, 5β-dihydrotestosterone, 11-ketotestosterone, estrone, 17β-estradiol, 17α-hydroxyprogesterone, 17α,20β-dihydroxy-4-pregnen-3-one, 5β-pregnane-3α,17α,20α-triol, 5β-androstane-3α,17β-diol, 5β-androstane-3β,17β-diol, 5β-pregnane-3α,17α,20β-triol, 5β-androstane-3α,11β-diol-17-one, 5β-pregnane-3α,17α-diol-20-one, 17α,20β,21-trihydroxy-pregn-4-en-3-one and 11-deoxycortisol in water was developed using gas chromatography-mass spectrometry with selected ion monitoring. Fish sex steroids were extracted from holding water using C18 SPE cartridges, and eluted using methanol. Free and glucuronide conjugate levels of steroids were determined, glucuronide conjugates by hydrolysis using β-glucuronidase prior to derivatisation and free steroids by direct derivatisation. The method thus developed was applied to twelve goldfish holding water samples. Androstenedione was found to be excreted in considerably higher quantities by female ovulatory goldfish than spermiating goldfish, in contrast to previous research in this area.