Chemiluminescent assays for /3-D-galactosidase and alkaline phosphatase are described which use novel chemiluminogenic substrates, o-aminophthalylhydrazide-f3-D-galactoside and o-aminophthalylhydrazide-0-phosphate, respectively. Each substrate is enzymatically hydrolyzed under the optimum conditions to release luminol, which luminesces in the presence of potassium hexacyanoferrate(III) in an alkaline medium. The detection limits (S/N=3) of the enzyme activities (µU per tube) were 50 for 13-D-galactosidase and 100 for alkaline phosphatase.