D-Luciferin acetate contains an aceate moiety attached at the 6-O-position, thus preventing it be recognized by the firefly luciferase enzyme. However, the acatate moiety is removed effectively by esterase activity, and the resulted D-luciferin is well recognized by luciferase. D-Luciferin acetate is a sensitive substrate for the chemiluminescent measurement of esterase activity in homogeneous assays, or in cell lysate samples in combination with luciferase and its cofactors.