Context: Short residence time, poor bioavailability and poor permeability are the major problems for conventional eye drops treatment. Objective: The aim of this article is to develop, optimize and ex vivo–in vivo investigation of brimonidine tartrate in situ gel as compared to marketed eye drops for the treatment of glaucoma. Materials and methods: The effect of independent variables, namely concentrations of polymers, on various dependent variables like viscosity at physiological pH and in vitro drug release were studied by using 32 factorial design. Further the optimized formulation was characterized for ex vivo and in vivo study. Results and discussion: Experimental data demonstrated that optimized in situ gel formulation (F8) showed in vitro–ex vivo sustained release profile with polymer composites carbopol 974P and HPMC K4M. After 5 h of ex vivo transcorneal permeation study, the amount recovered from the corneal surface on the donor chamber 12.40% (124 ug) and the amount collected from the receptor chamber 76.8% (760 ug) of the initial dose 1 mg. The total amount recovered from the permeation experiment was 89.2%. Bioadhesive carbopol 974P and viscosity HPMC K4M composites optimized formulation (F 8) produce greater influence on the duration of drug action and improved intraocular pressure reduction activity as compared to marketed eye drop solution in in vivo study. Conclusion: The developed in situ gelling system as a promising ophthalmic formulation to prolong the drug lowering effect on the intraocular pressure.