The phytohormone (+)-7-iso-jasmonoyl-l-isoleucine (JA-Ile) causes protein–protein interactions (PPI) between F-box Protein CORONATINE INSENSITIVE 1 (COI1) and JASMONATE ZIM DOMAIN (JAZ) transcriptional repressor. A total of 13 JAZ subtypes are encoded in the genome of Arabidopsis thaliana; however, their genetic redundancy obfuscates the individual function of each JAZ. One approach to decipher this redundant signaling network is chemical genetics, using small molecules specific to individual JAZ subtype, for which a reliable and high-throughput screening system of the ligands for all combinations of COI1–JAZs would be indispensable. In this chapter, we describe a fluorescence anisotropy-based quantitative screening system for the ligands of COI1–JAZ co-receptors. Our method is applicable to agonists and antagonists of the COI1–JAZs.