Time-resolved fluorescence resonance energy transfer, TR-FRET, is a time-gated fluorescence intensity measurement which defines the relative proximity of two biomolecules (e.g., proteins, peptides, or DNA) based on the extent of non-radiative energy transfer between two fluorophores with overlapping emission/excitation spectra. In these assays, an excited lanthanide ion acts as a “donor” that transfers energy to an “acceptor” fluorophore through dipole–dipole interactions. A FRET signal is reported as the ratio of acceptor to donor emission following donor excitation. When a donor-conjugated protein interacts with an acceptor-conjugated protein, the donor and acceptor fluorophores are brought in close proximity allowing energy transfer from the donor to the acceptor resulting in a FRET signal. Because the lanthanide donors have a long emission half-life, the energy transfer measurement can be time-gated, which dramatically reduces assay interference (due to background autofluorescence and direct acceptor excitation) and thereby increases data quality. Here, we describe a TR-FRET assay that monitors the interaction of the estrogen receptor (ER) α ligand binding domain (labeled with a terbium chelate via a streptavidin–biotin interaction) with a sequence of coactivator protein SRC3 (labeled directly with fluorescein) and the disruption of this interaction with a peptide and a small molecule inhibitor.