AIM: To determine moxonidine in aqueous humor and iris-ciliary body by reversed-phase high performance liquid chromatography (RP-HPLC), and to evaluate the retinal neuroprotective effect after topical administration with moxonidine in a high intraocular pressure (IOP) model. METHODS: The eyes of albino rabbits were administered topically and ipsilaterally with 0.2% moxonidine. A RP-HPLC method was employed for the identification and quantification of moxonidine between 2 and 480min, which presented in the aqueous humor and iris-ciliary body. Flash electroretinography (F-ERG) amplitude and superoxide dismutase (SOD) level were measured between day 1 and day 15 after topical administration with moxonidine in a rabbit model of high IOP. Histological and ultrastructural observation underwent to analyze the changes of retinal morphology, the inner retinal layers (IRL) thickness, and retinal ganglion cell (RGC) counting. RESULTS: Moxonidine was detectable between 2 and 480min after administration, and the peak concentration developed both in the two tissues at 30min, 0.51 μg/mL in aqueous humor and 1.03 μg/g in iris-ciliary body. In comparison to control, F-ERG b-wave amplitude in moxonidine eyes were significantly differences between day 3 and day 15 (P<0.01) in the high IOP model; SOD levels were significantly higher at all time-points (P<0.01) with a maximum level of 20.29 U/mgprot at day 15; and RGCs were significantly higher (P<0.05). CONCLUSION: Moxonidine is a viable neuroprotective agent with application to high IOP model. All layers of retina, including RGC layer, retinal nerve fiber layer and INL, are more preserved after moxonidine administration. SOD plays a neuroprotective role in ocular hypertension-mediated RGC death.