In this research work herbal drugextract of Gingko Biloba was used to prepare in-situ gel for treating glaucoma. Totalnumber of six formulations were prepared and evaluated for different evaluation parameters. The percent drug release for all formulations was carried out in Franz diffusion cell using simulated tear fluid the formulation, G5 showed a maximum release of 98.88 % in a total of 8 hours. The results of ex vivo studiesindicated that 82.68% of the drug permeated from the formulations, demonstrating a sustained effect. The ocular irritancy test conducted on the ocular in situ gel yielded an overall score of zero, indicating the absence of any signs of ocular irritation, such as redness, increased tear production, or swelling (edema). Also, the in-situ gel of Gingko Biloba Extract(G5) formulations demonstrated a good percentage reduction in IOP, with a value of 17.3±1.64 mmHg. The in-situ gel formulation achieved a Cmax of 8.9 mg/mL after 4 hours, which was 10 times higher than the Cmax of the timolol eye drop (0.908 mg/mL) obtained after 2 hours. This indicates that thein-situ gel formulationsachieved a significantly higher peak concentration in the aqueous humor compared to the timolol eye drop.