This chemical is widely used as a substrate in biochemical assays, particularly for the detection of β-glucuronidase enzyme activity. It is commonly employed in microbiology and molecular biology to identify and quantify bacterial colonies, especially Escherichia coli, which express β-glucuronidase. When the enzyme cleaves the substrate, it produces an insoluble blue-green indigo derivative, making it a valuable tool for visual or spectrophotometric detection. It is also utilized in histochemistry and immunohistochemistry for locating enzyme activity in tissue samples. Additionally, it plays a role in environmental monitoring to assess water quality by detecting fecal contamination. Its sensitivity and specificity make it a key component in diagnostic and research applications.