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Acetazolamide (ACZ), a carbonic anhydrase inhibitor, is employed to decrease the intraocular pressure (IOP) in glaucoma patients. ACZ is characterized by low aqueous solubility and reduced corneal permeation. The aim of the present work is to incorporate ACZ into bilosomes to achieve improved ocular delivery. ACZ bilosomes were prepared by the thin film hydration method using Span 60, cholesterol along with various bile salts (sodium cholate, sodium deoxycholate, sodium taurocholate and sodium tauroglycocholate) at two molar ratios (1:1:0.1 and 1:1:0.2). The obtained vesicles exhibited high entrapment efficiencies (69.03–74.24%), nanometric vesicle diameters (350–735 nm) with monodisperse distribution, zeta potential values < -43.4 and spherical morphology under transmission electron microscopy. In vitro drug release profiles revealed a biphasic release pattern governed by a Fickian diffusion …