Identification of ligands that interact with nuclear receptors is both a major biological problem and an important initial step in drug discovery. Several in vitro and in vivo techniques are commonly used to screen ligand candidates against nuclear receptors; however, none of the current assays allow screening without modification of either the protein and/or the ligand in a high-throughput fashion. Differential scanning fluorimetry (DSF) allows unmodified potential ligands to be screened as 10µL reactions in 96-well format against partially purified protein, revealing specific interactors. As a proof of principle, we used a commercially-available nuclear receptor ligand candidate chemical library to identify interactors of the human estrogen receptor α ligand binding domain (ERα LBD). Compounds that interact specifically with ERα LBD stabilize the protein and result in an elevation of the thermal denaturation point, as monitored by the environmentally-sensitive dye SYPRO orange. We successfully identified all three compounds in the library that have previously been identified to interact with ERα, with no false positive results.