A chemiluminescent substrate of hydrolytic enzyme having the following general Formula I, as follows: Lumi-M-P  Formula I where “Lumi” is a chemiluminescent moiety capable of producing light (a) by itself, (b) with MP attached and (c) with M attached. Examples of Lumi include chemiluminescent acridinium compounds, benzacridinium compounds, quinolinium compounds, isoquinolinium compounds, phenanthridinium compounds, and lucigenin compounds, spiroacridan compounds, luminol compounds and isoluminol compounds. M is a multivalent heteroatom having at least one lone pair of electrons selected from oxygen, nitrogen and sulfur, directly attached to the light emitting moiety of Lumi at one end and to P at the other end. P it a group that can be readily removed by hydrolytic enzymes. An enzymatic reaction utilizing the above compound is the following: where HE is a hydrolytic enzyme Lumi-M is a chemiluminescent product having physical and/or chemical properties different from those of Lumi-M-P.