The H₂O₂ Substrate reacts directly with H₂O₂ to create the Luciferin Precursor. The ROS-Glo™ Substrate Detection Solution converts the Luciferin Precursor to luciferin and also provides the Ultra-Glo™ Luciferase to produce light. Light signal is proportional to the amount of H₂O₂ in the sample well.

The assay follows a simple two-reagent-addition protocol that does not require sample manipulation. The ROS-Glo™ H₂O₂ Substrate can be added to assay wells with the treatment or at the end of the treatment incubation period, and data can be recorded in less than 2 hours after reagent addition.

HepG2 cells were treated with either ROS-generating compounds (menadione or pyrogallol) or a cytotoxicity inducing reagent (digitonin) and incubated at 37°C for 2 hours. CellTox™ Green Dye (membrane impermeable DNA binding dye as an indicator of cytotoxicity) and H₂O₂ Substrate were added at the time of dosing. After incubation fluorescence from the CellTox™ Green Dye was measured followed by addition of ROS-Glo™ Detection Solution. Luminescence signal from the ROS-Glo™ Assay was measured following 20‑minute incubation.