A series of four aliphatic carboxylic acid esters (acetate, caproate, laurate and palmitate) of 2-(4-hydroxyphenyl)-4,5-diphenylimidazole (HDPI) was prepared. Usefulness of these esters as fluorescent substrates for the measurement of lipase activity was evaluated using a high performance liquid chromatographic method. The hydrolysate, HDPI, could be well separated from each substrate by a reversed-phase column and detected at 440 nm emission (cutoff-filter) with 360 nm excitation. Lipase from Candida cylindracea was used for the investigation. From their kinetic parameters, the caproate and laurate were found to be suitable for the lipase. Using the laurate, from 5 to 500 mU per assay tube of the lipase was determined.