Glaucoma is an ocular disease condition that is more frequent in older persons and causes high intraocular pressure, posing a major danger to vision if not treated. Because the marketed brinzolamide has disadvantages, the present optimal formulation was developed with our preservatives. The current investigation was aimed to ensure the safety and effectiveness of created formulation for ocular medication administration. The prepared 0.5% Brinzolamide ophthalmic solution 0.5% was tested in New Zealand White rabbits well tolerated and reduced intraocular pressure (IOP) 11.11 mmHg compared with saline and placebo control samples. Additionally, a significant increase in the area under change in IOP from baseline (ΔIOP) vs. time curve and a longer mean residence time (MRT) were also observed for the test formulation compared to the commercially available Brinzolamide 1% and p < 0.001. The T/R ratio was calculated for the pharmacokinetic parameters of Cmax, Tmax, AUC0-t and MRT cumulatively for left and right eyes. The blood concentration versus time data shown the similar exposure for both test and reference formulations. The T/R ratio of blood for Cmax, Tmax, AUC0-t and MRT was found to be within the acceptable bioequivalence range which shows the equivalence of Test formulation with that of Reference formulation.