It is commonly used as a substrate with hydrogen peroxide for a peroxidase enzyme or alone with a laccase enzyme. Its use allows the reaction kinetics of peroxidases themselves to be followed. In this way it also can be used to indirectly follow the reaction kinetics of any hydrogen peroxide producing enzyme, or to simply quantify the amount of hydrogen peroxide in a sample.

$ABTS\; +\; H\_2O\_2\; overrightarrow\{\_\{HRP\}\}\; ABTS^+\; +\; H\_2O$

This compound is chosen because the enzyme facilitates the reaction with hydrogen peroxide, turning it into a green and soluble end-product. Its new absorbance maximum of 405 nm light can easily be followed with a spectrophotometer, a common laboratory instrument. It is sometimes used as part of a glucose estimating reagent when finding glucose concentrations of solutions such as blood serum.