The applications of ozone are not only restricted to environmental remediation or industrial areas. This gas has been applied in medicine to treat several diseases, where positive effects have been confirmed by many clinical studies. According to the European Medical Society of Ozone and the National Center of Scientific Investigation in Cuba, it has not been possible to validate ozone's effectiveness by traditional analytical methods. Thus, this investigation proposed evaluating the effect that ozone has on biological substrates (murine models with induced carcinogenic tumors, inflammation, and wounds), studying the variations that ozone (dissolved in physiological solution or ozonated vegetable oils) provokes over the total unsaturation of lipids (TUL), and by using the so-called method double bond index (DB-index), make a correlation with the dynamic reactions obtained by several analytical methods according to each experimental stage considered in this study.