The complex structure of the eye poses challenges in delivering drugs effectively, which can be circumvented by employing nanotechnologies. The present study aimed to prepareacetazolamide-loadedleciplex (ACZ − LP) using a simple one-step fabrication approach followed byoptimization employing a 32 Full Factorial Design. The ACZ – LP demonstrated high entrapment efficiency (93.25 ± 2.32 %), average diameter was recorded around 171.03 ± 3.32 with monodisperse size distribution and zeta potential of 41.33 ± 2.10 mV. Invitro release and ex vivo permeation studies of prepared formulation demonstrated an initial burst release in 1 h followed by sustained release pattern as compared to plain acetazolamide solution. Moreover, an ex vivo corneal drug retention (27.05 ± 1.20 %) and in vitro mucoadhesive studies with different concentration of mucin indicated strong electrostatic bonding confirming the mucoadhesive characteristics of the formulation. Additionally, the histopathological studies ensured that the formulation was non-irritant and nontoxic while and HET-CAM ensured substantial tolerability of the formulation. The in vivo pharmacodynamic investigation carried out on a rabbit model demonstrated that treatment with ACZ − LP resulted in a significant and prolonged reduction in intraocular pressure as compared to plain acetazolamide solution, acetazolamide oral tablet, and Brinzox®. In summary, the ACZ − LP is anefficient and versatile drug delivery approach which demonstrates significant potential in controlling glaucoma.