Estrogens are released to the aquatic environment by wastewater treatment plant (WWTP) effluents and can affect wildlife. In the last three decades, many in vitro assay platforms have been developed to detect and quantify estrogenicity in water. In 2018, the International Organization for Standardization (ISO) standardized protocols became available for three types of in vitro estrogen receptor transactivation assays (ERTAs) detecting estrogenicity in 96-well plates (ISO19040 1-3). Two ERTAs—lyticase Yeast Estrogen Screen (L-YES) and Arxula YES (A-YES)—use genetically modified yeast strains, whereas the third utilizes stably transfected human cells. One human cell based assay is ERα-CALUX, which is based on a genetically modified human bone osteosarcoma cell line. In the present study, we characterized the performance, comparability, and effectiveness of these three ERTAs, including an evaluation involving proposed water quality thresholds (effect-based trigger values [EBTs]). For a robust evaluation, we collected 52 effluent samples over three sampling campaigns at 15 different WWTPs in Switzerland. Estrogen receptor transactivation assay results were correlated and compared with results from chemical analysis targeting known estrogens. The three ERTAs showed comparable data over all campaigns. However, the selection of EBTs plays a significant role in the interpretation and comparison of bioassay results to distinguish between acceptable and unacceptable water quality. Applying a fixed cross-assay EBT for effluent of 4 ng L⁻¹ resulted in varying numbers of threshold exceedances ranging between zero and four samples depending on the ERTA used. Using assay-specific EBTs showed exceedances in eight samples (ERα-CALUX) and in one sample (A-YES), respectively. Thus, proposed EBTs do not produce similar risk profiles across samples and further refinement of assay-specific EBTs is needed to account for assay-specific differences and to enable the application of ERTAs as effect-based methods in environmental monitoring. Environ Toxicol Chem 2022;41:2512–2526.