Biosensors developed in yeast cells represent attractive research area in biomedicine because they allow for detection of molecules of various structures and biological activities, economically and simply, without use of harmful radioactive materials. We have focused our attention on identification of androgen, glucocorticoid and estrogen receptor α/β ligands using fluorescent biosensors in yeast. Identification of compounds that modulate the activity of androgen (AR) or estrogen receptors (ER) is one of the major goals in the design of new treatments of hormone-dependent cancers. Similarly, glucocorticoid receptor (GR) ligands are used to treat autoimmune and inflammatory diseases, but due to a large number of side effects and drug resistance, great effort has been made to find new modulators. In this study, ligand binding domains (LBDs) of AR, ERα, ERβ or GR fused with yellow fluorescent protein (YFP) were expressed in Saccharomyces cerevisiae. Recombinant yeast cells were treated with tested steroid hormone or bile acid derivatives and, due to fluorescence resonance energy transfer phenomenon following ligand-binding, relative binding affinities were quantified fluorimetrically. Our results show that some of the tested compounds have moderate to high binding affinity for particular steroid receptors, similar to natural ligands, while affinities of other compounds were low or negligible. To elucidate mechanisms of action for these compounds, additional experiments are necessary, and to better understand the molecular interactions within the ligand-binding pocket of the receptor, molecular docking analysis can be conducted. In summary, the yeast-based biosensors used in this work have proven to be very useful for in vitro screening of novel anti-cancer and anti-inflamatory drug candidates, as well as for elimination of compounds that do not deserve further attention and resources due to their lack of desired bioactivities.