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The detection and quantification of radicals has useful chemical, biological, and physical applications. When certain radicals are produced in aqueous solutions, they react with water in the presence of dissolved oxygen, forming hydrogen peroxide. In order to quantify the concentration of these hydrogen peroxide molecules in a given sample, we can employ the Fenton reaction in which hydrogen peroxide oxidizes ferrous ions. When this reaction occurs in the presence of xylenol orange, the resulting ferric ion complexes with xylenol orange to induce a visible color change from yellow to brown. The stability and consistency of this method make it a reliable tool for the detection and quantification of hydrogen peroxide in aqueous solutions. Though the Fenton reaction is commonly used to detect hydrogen peroxide, its conditions have not been optimized to operate over a low concentration range. In this study, we optimize the component concentrations to achieve detection and quantification of hydrogen peroxide ranging in concentration from 0.2 μM to 20 μM. For these optimized component concentrations, we determined the reaction time of hydrogen peroxide under our specific conditions, and evaluated the stability of the solution both on its own and when reacted with hydrogen peroxide. Using these conditions, we used UV-visible spectroscopy to observe changes in absorbance measured at 440 nm and 580 nm that occur with increased concentration of hydrogen peroxide and correspond to peaks associated with unbound and complexed xylenol orange, respectively. We developed a calibration curve that uses a ratio of peak heights at 440 nm and 580 nm to assess hydrogen peroxide concentration. This assay and calibration curve enable the detection and quantification of hydrogen peroxide concentration, which will be used to measure radical concentrations generated from light- or force-induced bond cleavage in polymers, and has practical applications in the testing of biological and food samples.