D-Luciferin ethyl ester contains a methyl moiety attached at the carboxy position, thus preventing it be recognized by the firefly luciferase enzyme. However, the ester is hydrolyzed effectively by lipase activity, and the resulted D-luciferin is well recognized by luciferase. D-Luciferin methyl ester is a sensitive substrate for the chemiluminescent measurement of lipase activity in homogeneous assays, or in cell lysate samples in combination with luciferase and its cofactors.