Hepatocyte nuclear factor 4α (HNF4α) is a liver enriched transcription factor that belongs to the nuclear receptor family, and controls the expression of genes involved in various different functions, such as nutrient transport, metabolism, growth and differentiation. Recently, fatty acids and/or fatty acyl CoAs as well as some xenobiotics (e.g. hypolipidemic drugs) have been suggested as HNF4α ligands. The aim of this study was to identify potential ligands for HNF4α, using a DAUDA fluorescence displacement assay. LFABP was expressed and purified as a positive control, and used successfully to validate the DAUDA assay. Rat HNF4αLBD was successfully expressed and purified, therefore further work for ligand identification was performed using the rat HNF4α. Suitability of the DAUDA fluorescence displacement assay for screening of ligands for rat HNF4α was confirmed by a characteristic blue shift in fluorescence emission upon protein binding, as previously reported. Ligands screened in this study included five different fatty acids, palmitoyl CoA, and hypolipidemic xenobiotics of the fibrate family (fenofibrate, bezafibrate, clofibrate), as well as pioglitazone (a thionolidinedione).