This study aimed to provide irrefutable evidence of the preventive effects of oxymatine (OMT) on a model of endotoxin induced glaucoma in Wistar rats which can be attributed to its anti-inflammatory, antioxidant, and TNF-α antagonistic properties. To assess the impact of OMT on uveitic glaucoma, the normal group received 100 μL distilled water topically for 15 days, while the glaucoma control group was induced with uveitic glaucoma by applying 10 μL of 10 μg/mL lipopolysaccharide (LPS) topically for 3 consecutive days. The treatment groups were then given OMT solution at a volume of 50 μL with varying doses of 0.25%, 0.5%, and 1% once a day via topical administration for 15 days. In addition, as a standard, the animals were also given 100 μL of 1% dorzolamide topically for 15 days. All ophthalmic dosing was carried out by pulling the lower eye-lid of the experimental animals and administration of the respective solutions. The study uses cutting-edge real-time imaging of the retinal vasculature in anesthetized animals, postsacrifice lenticular picturization and biochemical evidence to support the changes in the retinal layers. LPS induced animals demonstrated increased IOP, disrupted antioxidant systems, massive lipid damage, enhanced TNF-α activity and changes in intracellular ATPase and ionic activities. The damaged retinal vasculature and lenticular opacification further supported the biochemical evidence. However, using OMT at a 1% dosage effectively enhanced the antioxidant levels, regulated intracellular ion concentration and ATPases, decreased TNF-α activity, and counteracted mechanobiological changes in the visual front and retina. Moreover, OMT can successfully normalize intraocular pressure, making it a highly beneficial treatment option for glaucoma., (© 2024 Wiley Periodicals LLC.)