Peroxisome proliferator-activated receptor γ (PPARγ) is a member of the nuclear receptor superfamily involved in insulin sensitization, atherosclerosis, inflammation, and carcinogenesis. PPARγ transcriptional activity is modulated by specific ligands that promote conformational changes allowing interaction with coactivators. Here we show that the fluorophore 1-anilinonaphthalene-8-sulfonic acid (ANS) binds to PPARγ–LBD (ligand binding domain), displaying negligible interaction with other nuclear receptors such as PPARα and retinoid X receptor α (RXRα). ANS binding is competed by PPARγ agonists such as rosiglitazone, 15-deoxy-Δ 12,14-prostaglandin J 2 (15d-PGJ 2), and 9,10-dihydro-15-deoxy-Δ 12,14-prostaglandin J 2 (CAY10410). Moreover, the affinity of PPARγ for these ligands, determined through ANS competition titrations, is within the range of that reported previously, thereby suggesting that ANS competition could be useful in the screening and characterization of novel PPARγ agonists. In contrast, gel-based competition assays showed limited performance with noncovalently bound ligands. We applied the ANS binding assay to characterize a biotinylated analog of 15d-PGJ 2 that does not activate PPAR in cells. We found that although this compound bound to PPARγ with low affinity, it failed to promote PPARγ interaction with a fluorescent SRC-1 peptide, indicating a lack of receptor activation. Therefore, combined approaches using ANS and fluorescent coactivator peptides to monitor PPARγ binding and interactions may provide valuable strategies to fully understand the role of PPARγ ligands.