Introduction: To develop and optimize viscous eye drop for dorzolamide hydrochloride and timolol maleate used in Glaucoma-the leading cause of irreversible blindness worldwide. Methods and Materials: 32 full factorial design was applied in formulation optimization to study the relationships between critical process parameters (concentration of HPMC K15 and Carbopol 934) and critical quality attributes, i.e. viscosity, mucoadhesion index and cumulative drug release for both the drugs. Further, the optimized formulation was identified through derringer's desirability approach of multicriteria decision making technique and was fully characterized in terms of molecular interactions (DSC and FTIR) as well as in vitro and in vivo biological properties, including biological sensitivity/irritation studies performed on rabbits. Moreover, in vivo ocular pharmacokinetic study was performed using developed bioanalytical RP-HPLC method having mobile phase phosphate buffer (pH 6.8): methanol: Acetonitrile, 45:45:10 v/v/v in rabbit tear fluid employing protein precipitating sample preparation technique. Results: Results revealed that optimized batch does not have any ocular irritancy on rabbit eye and shows increase in mean residence time compared to marketed formulation. Conclusion: Hence, the design planning methodology clearly showed its usefulness for optimizing the formulation and the formulated Viscous eye drop proved to be a promising formulation strategy for ophthalmic delivery of dorzolamide and timolol maleate making it a potential alternative for the marketed formulation.