The human estrogen receptors (hER) are members of the nuclear hormone receptor (NHR) superfamily and represent important drug targets for the pharmaceutical industry. Initially, ligand binding assays were used to identify novel ligands using receptors purified from native tissues. With the advent of molecular cloning techniques, cell-based transactivation assays have been the gold standard for many years of drug discovery. With the elucidation of the structural mechanisms underlying the activation of NHRs, cell-free assays with purified receptors have become important tools to directly assess different binding sites (e.g., the hormone binding site or the cofactor binding site). The available cell-free assays have so far facilitated the study of one binding site at a time. With the introduction of Terbium (Tb(3+))-based time-resolved fluorescence energy transfer (TR-FRET), it has become possible to measure 2 different interactions within 1 test tube in parallel. The authors have applied this technology to develop a dual readout system for the simultaneous monitoring of steroid hormone site binding and cofactor peptide recruitment. They took advantage of a commercially available fluorescent tracer as an indicator for classical steroid site binding and designed a novel peptide derived from the peroxisome proliferator-activated receptor gamma coactivator-1a (PGC1a) as an indicator for functional agonistic behavior of a test compound. The established assay is able to differentiate between agonists, antagonists, partial agonists, and compounds binding to the cofactor recruitment site. The IC(50) values obtained for a number of reference compounds in the multiplexed assay are in concordance with published data. The simple 1-step mix-and-measure protocol gives excellent quality and robustness and can be miniaturized to 5-microL volume.