The peroxisome proliferator-activated receptor gamma (PPARγ) is the target for the thiazolidinedione class of potent insulin-sensitizing drugs, which includes rosiglitazone and pioglitazone. However, their usage has been restricted due to severe side effects. Recent data have shown that specifically inhibiting the cyclin-dependent kinase 5 (Cdk5)-mediated phosphorylation of PPARγ at Ser273 may lead to novel insulin sensitizers with fewer side effects. Here we describe a novel enzyme-linked immunosorbent assay (ELISA) in the 384-well format, which enables screening for PPARγ ligands that inhibit phosphorylation at Ser273 by Cdk5. The assay is robust with a Z-factor > 0.6, demonstrating its suitability for high-throughput screening. We demonstrate the suitability of this assay for profiling of published PPARγ ligands and identification of novel compounds that prevent the Cdk5-mediated phosphorylation of PPARγ at Ser273 in a 622 compound pilot study. Our assay enables the discovery and development of novel therapeutic agents for use in type-2 diabetes. Furthermore, our results in combination with structural analysis of reported PPARγ ligand binding domain X-ray structures give a molecular rationale for the Cdk5-mediated phosphorylation of PPARγ at Ser273.