Previously, we reported the development of two in vitro time-resolved Föster resonance energy transfer (tr-FRET)-based assays for evaluating the potency and efficacy of different ligands of thyroid hormone receptor (TR) for regulating the recruitment of coregulators. We could measure independently, in separate assays, both the recruitment of SRC3 (steroid receptor coactivator 3, a transcriptional coactivator) and the dissociation of NCoR (nuclear receptor corepressor, a transcriptional corepressor) from a TR*retinoid X receptor (RXR) heterodimer bound to a DR+4 thyroid hormone response element (TRE). Here, by using the distinct emission peaks of Tb(3+), the donor fluorophore used to label the TRE-bound TR*RXR heterodimers, and selecting two distinct acceptor fluorophores, fluorescein and cyanine 5, to label of NCoR and SRC3, respectively, we have integrated our previous two assay formats into a single assay. Thus, we can measure the potency of TR ligands simultaneously for NCoR dissociation and SRC3 recruitment activities in a system that mimics many features of the cellular context of TR action. The performance of this dual assay was tested with a known, highly potent physiological TR ligand, triiodothyronine (T(3)), and with a synthetic TR antagonist, NH-3. Measured potencies and efficacies of these two TR ligands from this dual assay are highly comparable to those obtained from the two independent assays. Thus, this dual-acceptor tr-FRET assay further simplifies the measurement of ligand-modulated TR-coregulator interactions and should improve the overall efficiency of the screening process of TR drug discovery programs.