ABSTRACTThe vitreous humor undergoes liquefaction with age, resulting in complications that may require a vitrectomy, or surgical removal of the vitreous from the eye. Silicone oil, a common vitreous substitute, lacks properties similar to the natural vitreous. In particular, it lacks antioxidants that may be necessary to reduce oxidative stress in the eye. The purpose of this study was to evaluate antioxidant‐loaded hydrogel vitreous substitutes in a pilot in vivo study. Ascorbic acid and glutathione were loaded into synthesized PEGDA hydrogels. Following vitrectomy, experimental antioxidant hydrogels or silicone oil were injected into one eye of rabbits, while the other eye served as untreated or sham control. Ophthalmic assessments, including electroretinography, were performed. Levels of glutathione and ascorbic acid were higher in the eyes treated with the antioxidant‐loaded hydrogel vitreous substitute, although this was not found to be significant after 28 days. There were no statistically significant differences between groups with respect to clinical examination, and ocular health scores, electroretinograms, and histology were normal. These results indicate minimal concerns for the hydrogel formulation or high levels of antioxidants. Future research will assess the capability of vitreous substitutes to prolong antioxidant release, with the goal of minimizing cataract after vitrectomy.