Luciferin is a substrate for the enzyme firefly luciferase.

D-luciferin potassium salt is the more commonly water soluble substrate for cell-based and in vivo reporter assays. D-luciferin sodium salt is an alternative salt form with higher water solubility. D-luciferin free acid is soluble in methanol or DMSO, but only sparingly soluble in water. DMNPE-caged D-luciferin is a D-luciferin ester derivative which can cross cell membranes efficiently. Once inside the cells, the ester is continuously hydrolysed to a supply of D-luciferin. Alternatively, a burst of D-luciferin release can be generated by UV photolysis.

Luciferase is encoded by the luc gene, which is widely used as a reporter gene. Luminescence at 560 nm is generated when luciferase catalyses the oxidation of luciferin by an ATP-dependent process. Cells and tissues have very low intrinsic bioiluminescence, allowing for highly sensitive detection of lucifease expression. In addition, luciferin/luciferase can be used for highly sensitive measurements of ATP levels.