Abstract: The subject matter disclosed and claimed herein concerns measuring the binding affinity of glucokinase (“GK”) using a fluorescence polarization (“FP”) assay. The FP method includes use of modified GK ligands bound to a fluorescent label. Binding affinity is determined by measuring displacement of fluorescent ligand by the known or suspected GK ligands. The subject matter disclosed and claimed herein provides a robust high-throughput FP assay for the determination of binding affinity of ligands to glucokinase. The FP binding assay displayed both glucose and nucleotide dependence, and a useful dynamic range. The binding IC50 data correlated well with GK activation EC50 data.