To evaluate the neuroprotective potential of drug candidates to treat human glaucoma, a short-term rodent model of retinal ganglion cell death was employed. Transient ischemia applied to the rodent retina, with subsequent reperfusion for 1–4 weeks, produces an experimental retinal ganglion cell death that is quantifiable. A widely used method to detect viable retinal ganglion cells involves surgical injection of labeling compounds into the superior colliculus of the rodent brain, the retrograde transport of the compounds along the axons to the retina, and subsequent microscopic evaluation of the retina. In order to circumvent the labor intensive and invasive surgery of this method, we sought an alternative means of assessing retinal ganglion cell survival that would be more suitable for high-throughput analysis. We therefore developed a method of immunolabeling whole retinas ex vivo with an antibody to Brn-3b, an antigen expressed in a subpopulation of retinal ganglion cells, that allows for detection of a representative retinal ganglion cell population. Fluorescently tagged Brn-3b immunolabeled retinas were flat-mounted, digitally imaged, and assessed using image analysis software. We determined that 60 min of ischemia caused a 49% and a 32% decrease in Brn-3b positive retinal ganglion cells in Lewis rats after 4 weeks reperfusion, and Sprague–Dawley rats after 2 weeks reperfusion, respectively. In Swiss Webster ND4 mouse retinas subjected to 45 min ischemia and 7 days reperfusion, we found a 70% decrease in Brn-3b positive cells. Thus, ex vivo immunolabeling of retinal ganglion cells using antibody to Brn-3b provides an alternative to other methods of quantifying retinal ganglion cells.