The ATP bioluminescence assay is based on the firefly luciferase enzymatic reaction and works by exposing a sample of cells to the enzyme luciferase and the substrate luciferin.

Luciferase is an enzyme that produces light or luminescence when it oxidizes its substrate. This reaction requires energy derived from ATP to proceed. Because only living cells in the exposed sample would produce a bioluminescent signal, providing researchers with valuable insight into cellular metabolism and overall cellular health. It also allows researchers to quantify the amount of living or dead cells within a sample based on the presence or absence of ATP.