Background: Glaucoma is widely treated using eye drops, but around 95% of the drug is lost by the ocular barrier resulting in low bioavailability. The incorporation of polymeric nanoparticles into mucoadhesive polymer containing in situ gel is generally helpful in the retention of nanoparticles on the eye and improves the efficacy of the formulation Objective: The objective of the present investigation has to develop polymeric brinzolamide (BRZ) nanoparticles laden with timolol maleate (TM) in situ gel formulation. Methods: The optimized BRZ nanoparticles were prepared using PLGA by nanoprecipitation technique utilizing 32 full factorial designs (FFD). Healthy New Zealand White Rabbit (250-300 g) was used for the pharmacokinetic and pharmacodynamic study. Design of the experiment was applied to optimize formulation and validate the model. Some evaluation parameters related to BRZ nanoparticles as well as in-situ gel, have been done Results: The results of FFD reveal that the optimized condition for drugs to polymer ratio (1:7) containing 0.98 %w/v for poloxamer 188 results in higher entrapment efficiency and drug release with 156.7 nm particle size. The in-situ gel formulation has been prepared using Gelrite (0.5%w/v), and HPMC K4M (0.5%w/v) shows acceptable results with sustained drug release up to 6±0.1 h. The rabbit model's in-vivo pharmacokinetics and pharmacodynamic data showed sustained release of drugs longer than the marketed formulation. Conclusion: The proposed formulation could successfully deliver therapeutic concentrations in the eye with prolonged resident time and serve as a potential alternative for the treatment of glaucoma.