The Cholesterol/Cholesterol Ester-Glo™ Assay measures cholesterol using a cholesterol dehydrogenase that links the presence of cholesterol to the production of NADH and the activation of a proluciferin that produces light with luciferase. In the presence of NADH, Reductase enzymatically reduces a proluciferin Reductase Substrate to luciferin. Luciferin is detected in a luciferase reaction using Ultra-Glo™ Luciferase and ATP, and the amount of light produced is proportional to the amount of cholesterol in the sample.

For measuring Cholesterol Ester, the assay includes a cholesterol esterase that will remove the fatty acid from cholesterol esters to produce one molecule of cholesterol per molecule of ester. The amount of cholesterol esters is determined from the difference of cholesterol measured in the absence (free cholesterol) and presence (total cholesterol) of esterase.