This article aims to describe a preclinical proof of concept for a novel once-a-day (OD) brimonidine ophthalmic nanosuspension. The preclinical proof of concept was established using New Zealand white rabbits as animal models. Dose-finding, multiple-dose efficacy, ocular pharmacokinetic, and hemodynamic studies were performed in normotensive rabbits. Steroid-induced ocular hypertension model in rabbits was used to study efficacy in glaucomatous pathophysiology. The test (0.35% OD suspension) and reference (0.15% three times a day [TID] solution) were compared. The intraocular pressure (IOP) reduction was sustained for 0.35% and 0.5% strengths but not for other lower strengths tested or reference strengths. A 0.35% OD suspension reduced IOP >2 mmHg after 24 h of dosing, which was not seen with the reference. After multiple dosing, 0.35% OD suspension reduced IOP by 4-6 mmHg after 24 h, which was comparable to the 0.15% TID reference solution. An ocular pharmacokinetic study showed that the brimonidine was rapidly absorbed and distributed throughout the eye after topical administration. Concentration was higher in tissues with high α receptors, such as cornea-conjunctiva, iris/ciliary body, and choroid/retina. The steady-state concentrations in these organs were also significant after 24 h of the last dose. There was an indication of increased plasma levels, so a hemodynamic study was performed to assess any adverse effects. All hemodynamic parameters were normal and no new unusual safety findings were observed. The study demonstrated that the novel brimonidine 0.35% ophthalmic nanosuspension is both safe and effective when administered OD and is comparable to the marketed reference formulation administered TID.