Abstract: A method is provided for determining the activity of an enzyme which releases methylumbelliferone (MU) from an MU-containing substrate wherein the enzyme has a pH optimum below the pKa of MU comprising: contacting a sample suspected of containing the enzyme with the MU-containing substrate at a pH suitable for activity of the enzyme to allow release of MU by the enzyme; contacting the sample with light of a wavelength in the range of about 310 nm to about 350 nm; determining fluorescence produced by the released MU, thereby determining the activity of the enzyme. This real time method provides improved diagnostic methods, for example for diseases associated with an abnormal level of activity of a glycosidase. The real time assay also can be used to screen compounds for their ability to modulate enzyme activity using MU-containing substrates.