Peroxisome proliferator-activated receptors (PPARs) and other members of the nuclear hormone receptor family are important drug targets for the treatment of metabolic diseases. PPAR α and PPAR γ play crucial roles in lipid and glucose metabolism, respectively. Therefore, screening methods that help to rapidly identify activators of these receptors should be of considerable value. A homogeneous fluorescence polarization (FP) ligand binding assay capable of rapidly identifying ligands that bind to both PPAR α and PPAR γ has been developed using purified PPAR α or PPAR γ ligand binding domains and a fluorescein-labeled analog (FLA) of a potent dual PPAR α/ γ activator. FLA activator showed good binding affinity toward both PPAR α ( K i = 0.7 μM) and PPAR γ ( K i = 0.4 μM). The binding of FLA activator was rapid and reached a plateau within 10 min. The resulting FP signal was stable for at least 18 h. The FP binding assay performed robustly in a 384-well format, and the average Z′ value was 0.77. There was a good correlation between the binding potency (IC 50 values) and rank order of binding potency for a panel of standard PPAR ligands obtained in FP binding assay and scintillation proximity assay or gel filtration binding assays using 3H-labeled PPAR α ( r 2 = 0.99) and PPAR γ ( r 2 = 0.99) ligands. There was also a good correlation of IC 50 values obtained by FP binding assay and scintillation proximity assay for the clinically used PPAR activators. Thus, the FP binding assay with a single fluorescein-labeled PPAR α/ γ dual activator offers a homogeneous nonradioactive, sensitive, robust, and less expensive high-throughput assay for detecting compounds that bind to both PPAR γ and PPAR α. Using this FP binding assay, we have identified a large number of PPAR α/ γ dual activators. A similar assay platform may be easily adapted to other members of the nuclear hormone receptor family.