This guidance document describes an in vivo screening assay where sexually mature female sticklebacks are exposed to suspected anti-androgenic chemicals during a limited part of their life-cycle (21 days). The AFFS can detect both androgens and anti-androgens; however its usefulness is greater in the detection of androgen antagonists. The protocol does not require an in situ pre-exposure period and measures one biomarker endpoint as indicator of (anti)androgenic activity, the level of spiggin in the female stickleback kidneys. Other measurements include survival and body weight; these are not considered endpoints in the AFSS but are needed to confirm statistical robustness of the assay (survival) and biomarker level (spiggin/g body weight).