We describe a simple fluorescence displacement assay to measure hydrolysis of arachidonoyl ethanolamide and oleoyl amide, two important pharmacological compounds. Hydrolysis at the amide linkage of these ligands releases a fatty acid as one of the products. The displacement of a fluorescent fatty acid analogue from rat liver fatty acid-binding protein by the released fatty acid can thus be measured as a decrease in fluorescence. This process is time- and concentration-dependent and shows hyperbolic enzyme kinetics. Electrospray ionisation mass spectrometry was used to validate the assay.