Using the EnzChek Phosphatase Assay Kit continuous monitoring of phosphatase activity, even at neutral or acidic pH, can be achieved. The kit contains Molecular Probes' patented 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) substrate (D6567). DiFMUP is about 100 times more sensitive than 4-methylumbelliferyl phosphate (MUP) for the detection of prostatic acid phosphatase at pH 5.5. The kit is perfect for the continuous assay of prostatic acid phosphatase, protein phosphatase 1 or almost any other phosphatase that can be assayed with nonprotein-based substrates such as MUP or 4-nitrophenyl phosphate (PNPP). Each kit contains a sufficient amount of substrate for ∼1000 assays, using a reaction volume of 100 μL per assay. Fluorescence (excitation/emission maxima ∼358/455 nm) can be measured in a fluorescence microplate reader or a standard fluorometer.