Steroid hormone receptors represent a major target in drug discovery. As ligand inducible transcription factors, their activity can be modulated by small lipophilic molecules. Here we describe two panels of potent and selective luciferase reporter cell lines based on cells with low endogenous steroid receptor activity (U2OS). The panels contain reporter cell lines for estrogen receptors α and β, androgen, glucocorticoid, mineralocorticoid, and progesterone receptors. In the first panel, the activation of either synthetic, steroid response elements containing promoter or viral promoter is mediated by full-length steroid receptors. The second panel is based on the expression of the chimeric receptor, which was created by the replacement of the N-terminal part of the molecule by Gal4 DBD and that binds to multiple UAS sites in the reporter promoter. Both panels were extensively characterized by profiling 28 ligands in dose response manner in agonist and antagonist mode. We have analyzed and compared the responses to tested ligands from both panels and concluded that in general both systems generated similar qualitative response in terms of potency, efficacy, partial agonism/antagonism, mixed agonistic/antagonistic profiles and the rank of potencies was well conserved between both panels. However, we have also identified some artifacts introduced by the Gal4/LBD reporter assays in contrast to their full-length receptor reporter counterparts. Keeping in mind the advantages and drawbacks of each reporter format, these cell lines represent powerful and selective tools for profiling large compound libraries (HTS) and for detailed study of mechanisms by which compounds exert their biological effects.