The Cholesterol Assay Kit utilizes a series of enzyme reactions to measure cholesterol levels in samples. First, cholesterol esterase is used to hydrolyze cholesterol esters into free cholesterol. Then, cholesterol oxidase is used to convert cholesterol into cholest-4-en-3-one and hydrogen peroxide. The cholesterol level in the sample is determined by quantifying the hydrogen peroxide generated by the enzyme reaction with a fluorogenic probe that can be measured with a spectrophotometer.