Histamine is a well-known mediator of allergic, inflammatory, and neurological responses. More recent studies suggest a role for histamine and its receptors in a wide range of biological processes, including T-cell maturation and bone remodeling. Histamine serum levels are regulated mainly by the activity of the histamine-synthesizing enzyme histidine decarboxylase (HDC). Despite the importance of this enzyme in many physiological processes, very few potent HDC inhibitors have been identified. HDC assays suitable for high-throughput screening have not been reported. The authors describe the development of a fluorescence polarization assay to measure HDC enzymatic activity. They used a fluorescein-histamine probe that binds with high affinity to an antihistamine antibody for detection. Importantly, they show that probe binding is fully competed by histamine, but no competition by the HDC substrate histidine was observed. The automated assay was performed in a total volume of 60 muL, had an assay window of 80 to 100 mP, and had a Z' factor of 0.6 to 0.7. This assay provides new tools to study HDC activity and pharmacological modulation of histamine levels.