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Fluorescence polarization is a valuable technique for characterizing small molecule inhibitors. Competition fluorescence polarization assays involve the displacement of a fluorescent ligand from the enzyme with increasing amounts of a competing compound. The technique allows the determination of inhibitor dissociation constants (Ki values) and evaluation of whether the data fit is consistent with competitive and stoichiometric binding, which enables the potency of small molecule inhibitors to be ranked. Fluorescence polarization assays have the advantage of being robust, non-radioactive, and formatted for 384-well plates. Inhibitor dissociation constants can be obtained without the necessity of separating bound and unbound species. Here we describe the development and characterization of a fluorescent ligand for the Aggrecanase-1 and Aggrecanase-2 enzymes, and present measured competition IC50 values and Ki values for hit compounds in the Aggrecanase program that aided the team in lead optimization.