In the past two decades much research effort has focused on the occurrence, effects, and risks of estrogenic compounds. However, increasing emissions of new emerging compounds may also affect the action of hormonal pathways other than the estrogenic hormonal axis. Recently, a suite of novel CALUX bioassays has become available that enables looking further than estrogenic effects only. By employing these bioassays, we recently showed high glucocorticogenic activity in wastewaters collected at various sites in The Netherlands. However, since bioassays provide an integrated biological response, the identity of the responsible biological compounds remained unknown. Therefore, our current objective was to elucidate the chemical composition of the wastewater extracts used in our previous study by means of LC-high-resolution Orbitrap MS/MS and to determine if the compounds quantified could account for the observed glucocorticoid responsive (GR) CALUX bioassay response. The mass spectrometric analysis revealed the presence of various glucocorticoids in the range of 13-1900 ng/L. In extracts of hospital wastewater-collected prior to sewage treatment-several glucocorticoids were identified (cortisol 275-301 ng/L, cortisone 381-472 ng/L, prednisone 117-545 ng/L, prednisolone 315-1918 ng/L, and triamcinolone acetonide 14-41 ng/L) which are used to treat a great number of human pathologies. A potency balance calculation based on the instrumental analyses and relative potencies (REPs) of the individual glucocorticoids supports the conclusion that triamcinolone acetonide (REP = 1.3), dexamethasone (REP = 1), and prednisolone (REP = 0.2) are the main contributors to the glucocorticogenic activity in the investigated wastewater extracts. The action of these compounds is concentration additive and the overall glucocorticogenic activity can be explained to a fairly large extent by their contribution.