Purpose: To evaluate the optimal therapeutic window of 7,8‐Dihydroxyflavone (DHF), an agonist of TrkB receptor, as a neuroprotectant for degenerative retinal ganglion cells (RGCs) due to an excitotoxic damage with N‐methyl‐D‐aspartic acid (NMDA). Methods: In adult albino Sprague–Dawley rats, NMDA (5 μl of 100 nM) was intravitreally injected into the left eye (day 0) and the animals were sacrificed after 7 days. In these animals, the treatment regimen of systemically administered DHF (5 mg/kg) was evaluated in four groups of increasing dose range (n = 8/group): a single injection of DHF (day −1), two injections (days −1 and 0), four injections (days −1, 0, 1 and 2) and seven injections (day −1 and one daily injection until processing). The number of Brn3a+RGCs was automatically quantified in whole‐mounted retinas. Once the optimal dose of DHF was stablished, the time course of Brn3a+RGCs degeneration was analysed at 3, 7 and 14 days after NMDA administration (n = 8/time‐point) and their treatment with 4 injections of DHF or vehicle (0.9% NaCl containing 1% DMSO). The number of Brn3a+RGCs in injured retinas was quantified and the spatial distribution was assessed with isodensity maps. As control, intact retinas were used. Results: Seven days after NMDA injury, DHF treatment increases the percentage of surviving Brn3a+RGCs from 27% to 68% when retinas were treated with one injection to four injections, respectively (p < 0.05). No significant changes were found between four or seven DHF injections, indicating that the optimal treatment regimen for DHF is four injections and showing a rescue of Brn3a+RGCs over the entire retinal surface. Using this DHF dose at different time‐points after NMDA injury, DHF treatment rescues most Brn3a+RGCs degeneration at 3 days and it decreases up to 56% at 14 days. Conclusions: DHF prevents RGC death following NMDA excitotoxic damage.