In this study, we assessed and compared the suitability of three in vitro screening tools for the measurement of estrogenic activity in sewage treatment plant effluents (STPEs). These assays were the yeast estrogen screen (YES), production of zona radiata proteins (ZRPs) in trout hepatocytes, and the induction of reporter gene expression in the transfected rainbow trout gonad cell line RTG-2. Data obtained with the YES were additionally compared with calculated estrogenicity, based on steroid analysis data of the effluents. For comparison purposes, the response of the in vitro systems toward the estrogenic chemicals β-estradiol, ethinyl estradiol, bisphenol—A, nonylphenol, and octylphenol was assessed. All three assays showed sensitivities in the same order of magnitude in response to the steroid compounds tested, with ZRP production being the least sensitive. Regarding the estrogenic environmental chemicals tested, the RTG-2 assay was more than an order of magnitude more sensitive than the other two assays. Despite their different sensitivities toward selected test chemicals, the three in vitro systems indicated estrogenic activity in the same concentration range for the tested STPEs. Calculated estrogenicity (chemical analysis) and measured estrogenicity (YES) were of the same order of magnitude for the STPEs tested. The present study indicates that all three in vitro systems, with the yeast-based system being the easiest and most robust, are applicable for the screening of estrogenic activity in effluent samples.