Ozone therapy is a widespread medical, veterinary, and dental procedure. The techniques are diverse and include the direct and controlled administration of ozone gas and the use of previously ozonated materials, as ozonized oils. Ozonation of oils alters the structure of fatty acids, generating peroxides, which are the agents in wound healing and has bactericidal effects. This work aimed to develop a chemiluminescence-based analytical technique for measuring the peroxidic content of ozonized oils. Sesame oil was ozonized at different time intervals, and the presence of peroxide characterized by FTIR and the peroxide content was determined by iodometry (Peroxide Index). The chemiluminescent analytical method was developed based on the oxidation of luminol and using horseradish peroxidase (HRP) as the catalyst. Figure 1 shows the light emission profile for a reaction in the presence and absence of ozonized oil. The studied parameters were: buffer type and pH, the concentrations of HRP and luminol, and the solvent used to dissolve the ozonized oil. The best conditions were: HRP 12.5 nM, luminol 0.5 mM, and Tris buffer 50 mM, pH 9.0. Figure 2 shows the correlation between the oil concentration and the total light emission. In conclusion, the method was sensitive and adequate to measure the oxidative potential of ozonized oils.