Abstract We developed a glaucoma-on-a-chip (GOC) model to evaluate the viability of retinal ganglion cells (RGCs) against high pressure and the potential effect of neuroprotection. A three-layered chip consisting of interconnecting microchannels and culture wells was designed based on simulation of physical parameters. The chip layers were fabricated from poly-methyl methacrylate sheets. Multiple inlet ports allow culture media and gas into the wells under elevated hydrostatic pressure (EHP). The bottom surface of the wells was modified by air plasma and coated with different membranes to model an extracellular microenvironment. The SH-SY5Y neuroblastoma cell line served as model cells to determine the best supporting membrane which was revealed to be PDL/laminin. Thereafter, the study experiments were performed using RGCs obtained from postnatal 5-7 Wistar rats purified by magnetic assisted cell sorting. Flow cytometry and immunocytochemistry assays demonstrated 70% purification for RGCs. The cultured RGCs were exposed to normal (15 mmHg) or elevated pressure (33 mmHg) for 6, 12, 24, 36 and 48 hours, with and without adding brain-derived neurotrophic factor (BDNF) or a novel BDNF mimetic (RNYK). RGC survival rates were 85, 78, 70, 67 and 61 percent under normal pressure versus 40, 22, 18, 12 and 10 percent under high pressure at 6, 12, 24, 36 and 48 hours, respectively (P <0.0001). BDNF and RNYK treatments induced separately an approximate two-fold decrease in the rate of RGC death under both normal and elevated pressures (p <0.01 to 0.0001). This GOC model recapitulated the effects of elevated pressure during relatively short time periods and demonstrated the neuroprotective effects of BDNF and RNYK.