Identification of chemicals with endocrine-disrupting activities in the past two decades has led to the need for sensitive assays for detection and monitoring of these activities in the environment. In vitro reporter gene assays represent a relatively fast and easy-to-perform method for detection of compounds that are able to bind to hormonal receptors and stimulate or silence their transactivation activity, thus interfering with the hormone signaling pathways. This paper reviews upgrades on reporter gene assays performed during the last decade. The utilization of new reporter genes (luciferase and green fluorescent protein coding genes) significantly improved the sensitivity of the tests and made them faster. Reporter gene assays now represent a high-throughput system for screening chemicals for hormonal activity. Finally, modification of test set-ups for testing anti-hormonal activities also enabled measurements of endocrine-disrupting activities in complex environmental samples such as sediments and wastewater treatment plant effluents.