Gestrinone is a synthetic steroid hormone with anti-estrogenic and anti-progesterone properties. It is used to treat endometriosis, shrink uterine fibroids and reduce menorrhagia; besides, it has been investigated for use as contraceptive. Also, due to its anabolic effects, it has been included in the banned list of performance enhanced drugs in sport. Polyclonal antibodies raised against bovine serum albumin coupled to gestrinone 3-carboxymethyloxime (3OCMO-G) were used to develop two highly sensitive and specific enzyme-linked immunosorbent assays for gestrinone. One of them, based on direct format, shows a detection limit (LD) of 0.09+/-0.03 ng L(-1). The second assay, hapten-protein coating format, can detect until (LD) 0.14+/-0.05 ng L(-1). Both immunoassays were also highly specific, showing negligible or no cross-reactivity to other anabolic steroids. The developed ELISAs detected lower amounts of gestrinone than those determined by the reference chromatographic HPLC/MS/MS methods. The direct format was applied to quantify this steroid in spiked human urine without sample pre-treatment, with recovery values between 76 and 122%.