Prostaglandin (PG) analogues may have an additional effect to protect neurons independent of IOP reduction. Only a few reports indicated that some PG analogues had neuroprotective effects or increased blood flow in in vivo and in vitro models. However, there is no comparative study using all clinically available PG analogues and also using primary culture of retinal ganglion cell (RGC). Our purpose of study is to investigate the direct neuroprotective effect of PG analogues on glutamate- and hypoxia-induced RGC death using rat purified primary RGC culture with latanoprost acid, travoprost acid, bimatoprost acid, bimatoprost, tafluprost acid, unoprostone, and PGF2α. Purified RGCs cultures were obtained from retinas of 6 days old Wistar rats, following a two-step immuno-panning procedure. After 72 h of cultivation, the neuroprotective effect of PG analogues (1 nM, 10 nM and 100 nM) was investigated by culturing the RGCs in 25 μM glutamate for a further 72 h or 5% O2 hypoxic condition for 24 h. The RGC viability under each condition normalized to that under normal condition without stress was evaluated as live cell percentage based on a total of 15 repeated experiments. As a result, 100 nM of latanoprost acid, tafluprost acid, bimatoprost acid, and bimatoprost significantly increased RGC survival rate by suppressing apoptosis. PG analogues indicated IOP independent neuroprotective effect on glutamate- or hypoxia-induced RGC death using rat primary RGC culture at clinically available intracameral concentration. Since those profiles were different from clinical efficacy in IOP reduction, the mechanism of neuroprotection may be not related to FP receptor stimulation.