D-Luciferin is widely used in bioluminescence imaging to monitor biological processes in live cells and small animal models. It serves as a substrate for the enzyme luciferase, producing visible light when oxidized, which allows researchers to track gene expression, tumor growth, and infection progression in real time. This application is especially valuable in preclinical studies, including cancer research, stem cell tracking, and vaccine development. D-Luciferin is also employed in ATP assays to measure cellular viability and microbial contamination, as the light output correlates directly with ATP levels. Its high sensitivity and low background noise make it ideal for in vivo imaging with minimal invasiveness.