Ecdysteroids are a group of steroid compounds present in many plant and invertebrate species. In arthropods, they function primarily as hormones involved in the regulation of molting. This protocol describes how to extract ecdysteroid hormones from ant specimens and subsequently quantify circulating levels of the hormone. The hormone can be extracted from hemolymph or from whole-body homogenates of insects and quantified by radioimmunoassay.