Encephalic vascular accident, or stroke, is the most common pathology of the central nervous system in humans. It is the second leading cause of death, as well as physical and cognitive disabilities, in developing countries. It is a vascular disorder that may present in an ischemic (more common) or hemorrhagic form. Ozone is a gas capable of oxidizing double bonds of organic molecules, thereby producing lipoperoxides and aldehydes. Stimulation of the immune and antioxidant system, and improvement in tissue vascularization and oxygenation, are few among several effects exerted by ozone. Ozone therapy has previously been shown to be effective in neuromodulation, neuroprotection, and nerve regeneration. The present study aimed to evaluate the effect of targeted mild ozone after inducing cerebral ischemia in vitro . The experiment was divided into two steps; in the first step, neuroblastoma lineage cells (SH-SY5Y) were subjected to 24 hours of hypoxia in an incubator culture chamber. Treatment with different concentrations of ozone (2–10 µg/mL), followed by an 2,5-diphenyl-2H-tetrazolium bromide assay, indicated a possible neuroregenerative effect at low concentrations. The same protocol was applied to canine amniotic membrane stem cells that were evaluated via colorimetric assay spectrophotometry, fluorescence microscopy, and flow cytometry. The metabolic conditions and cellular regeneration in cells at low ozone concentrations (3–8 µg/mL) correlated with lower levels of apoptosis and oxidative stress compared to cells not subjected to hypoxia. High concentrations of ozone (18–30 µg/mL) promoted an increase in rate of apoptosis and cell death. Therefore, we developed a novel protocol that mimics ozone therapy for ischemic stroke, using ozonized culture medium after hypoxia induction. Although more studies are needed to open new avenues for translational medicine, we conclude that ozone has a dose-dependent hormetic effect and can reverse the effect of ischemia in vitro at low concentrations.