Retinal ischemia, one of the most common cause of visual loss, is associated with blood flow inadequacy and subsequent tissue injury. In this setting, some treatments that can counteract glutamate increase, arouse interest in ischemic pathogenesis. Ketamine, a potent N-methyl-d-aspartate (NMDA) receptor antagonist, provides a neuroprotective pathway via decreasing the excitotoxicity triggered by excess glutamatergic. Thus, the goal of this study was to evaluate the safety of intravitreal use of ketamine and their potential protective effects on retinal cells in retinal ischemia/reperfusion model. Initially, ketamine toxicity was evaluated by cytotoxicity assay and Hen's egg chorioallantoic membrane (HET-CAM) method. Afterward, some ketamine concentrations were tested in rat's eyes to verify the safety of the intravitreal use. To investigate the neuroprotective effect on retinal, a single intravitreal injection of ketamine in concentrations of 0.059 mmol.L⁻¹ and 0.118 mmol.L⁻¹ was performed one day before the retinal injury by ischemia/reperfusion model. After 7 and 15 days, the retina activity was evaluated by electroretinogram (ERG) records and, lastly, by morphological analyzes. Cytotoxicity assay reveals that the maximum ketamine concentration that could reach retinal pigmented epithelium cells is 0.353 mmol.L⁻¹. HET-CAM assay showed that concentrations above 0.237 mmol.L⁻¹ are irritants to the eye. Thus, Ketamine in concentrations of 0.0237 mmol.L⁻¹, 0.118 mmol.L-1, and 0.059 mmol.L-1 were selected for in vivo toxicity test. ERG records reveal a tendency of b-wave amplitude to decrease as the luminous intensity increased, in the group receiving ketamine at 0.237 mmol.L⁻¹. Therefore, ketamine in concentrations at 0.059 mmol.L⁻¹ and 0.118 mmol.L-1 were chosen for the following tests. In the ischemia retinal degeneration model, pretreatment with ketamine was capable to promote a recovery of retinal electrophysiological function minimizing the ischemic effects. In histological analysis, the groups that received intravitreal ketamine showed a number of retinal cells significantly higher than the vehicle group. In TUNEL assay a reduction on TUNEL-positive cells was observed in all the layers for both concentrations which allow to affirm that ketamine contributes to reducing cell death in the retina. Transmission electron microscopy (TEM) reaffirms this finding. Ketamine intravitreal pretreatment showed reduced ultrastructural changes. Our findings demonstrate that ketamine is safe for intravitreal use in doses up to 0.118 mmol.L⁻¹. They seem to be particularly efficient to protect the retina from ischemic injury.