The glucocorticoid receptor (GR) belongs to the family of steroid receptors (SRs). These receptors regulate a vast selection of cell-, tissue-, and organism biology, and are also targets of endocrine disrupting chemicals warranting design and validation of in vitro assays. Here we report a “blinded” ring trial of an in vitro cell-based GR transactivation assay with four involved laboratories. The laboratories set up the assay and tested 34 selected chemicals with remarkably good concordance. There was agreement between all laboratories for the classification of activity in 97 % of the cases, and three or more laboratories were always in agreement. The within laboratory concordance was very high (99.6 %) with only one of all 272 triplicates deviating. The assay was, thus, deemed easily transferable and reproducible within and between laboratories, since they would arrive at the same qualitative results. Furthermore, for the chemicals with solid data regarding GR activation or inhibition, the laboratories arrived at the expected conclusion in all cases. Overall, the transfer and validation were successful, and the method is under evaluation to become an OECD test guideline. The method is expected to become valuable in tiered approaches for assessing chemicals or environmental samples together with other similar methods.