This study aims to evaluate the effect of different formulation variables (surfactant type and HLB value) adopting full factorial design (51. 21) using coacervation phase technique on in vitro characterization of dorzolamide hydrochloride (DZ)-loaded proniosomal gels, namely, entrapment efficiency percentage (EE%), vesicle size distribution, polydispersion index (PDI), and in vitro DZ release. The optimum formula F2 with a desirability value of 0.937 composed of 40mg DZ, 500mg span 60, 500mg of L-α-Lethicin, and 55.5mg cholesterol showing EE% of 84.5 ± 1.5%, PS of 189.5 ± 35.76nm with PDI 0.8 ± 0.28 and 58.51% ± 1.00 of DZ released after 8h was further evaluated using differential scanning calorimetry (DSC) and transmission electron microscopy (TEM). The effect of gamma sterilization on transcorneal permeation and stability of DZ from the selected formulation (F2) revealed that F2 was significantly tolerable, stable, and competent to corneal permeation confirmed by histological examination, confocal laser microscopy, and intraocular pressure (IOP) measurement. Significant corneal bioavailability was attained from formula F2 (370.6mg. h/m) compared to the market product Trusopt® eye drops (92.59mg. h/ml) following IOP measurement, thereby proniosomal gels could be considered as tolerable and competent ocular platforms for improving the transcorneal permeation of DZ.