DiFMUP is widely used as a fluorescent substrate in enzyme activity assays, particularly for detecting phosphatase enzymes such as alkaline phosphatase. When the enzyme cleaves DiFMUP, it produces a highly fluorescent product that can be easily measured using a fluorescence reader. This makes it ideal for high-throughput screening in drug discovery, diagnostic assays, and cellular imaging. Its low background fluorescence and high sensitivity allow for accurate detection of enzyme activity even at low concentrations. It is also employed in studies involving kinase pathways, where phosphatase activity needs to be monitored as part of signal transduction analysis.