The two Arxula adeninivorans-based bioassays (A-YGS and A-YGFS) described here provide sensitive and reliable screening for glucocorticoids in aquatic environments. The biocomponents of A-YGS and A-YGFS were constructed to carry the human glucocorticoid receptor (hGRα) and the phytase gene (phyK, derived from Klebsiella sp. ASR1) or a fluorescence dsRED gene (derived from the Discosoma sp.), as reporter genes. The responses of A-YGS and A-YGFS were measured photometrically and spectrofluorometrically respectively. The half effective concentration (EC50) and limit of detection (LoD) values for dexamethasone obtained with A-YGS and A-YGFS were 0.81 and 0.29μM, and 9.42 and 0.47μM, respectively. Furthermore, both bioassays exhibited different binding specificities for several natural and synthetic glucocorticoids: A-YGS – corticosterone>cyproterone acetate>mifepristone>dexamethasone>cortisol>methylprednisolone>prednisolone and A-YGFS – cortisol>corticosterone>dexamethasone>prednisolone>betamethasone>methylprednisolone. As proof of principle, A-YGS was used to assay total glucocorticoids in river water, wastewater influent and effluent taken from a wastewater treatment plant in Germany. Glucocorticoids were not detected in both A-YGS and LC–MS/MS in seven of the eight samples, however a dexamethasone equivalent (DEQ) was detected in an influent wastewater sample using A-YGS (0.055μM), while LC–MS/MS analysis showed that hydrocortisone and cortisone were present at 0.063 and 0.083nM, respectively.