The vitreous humor of the eye presents a crucial target for posterior segment therapy due to its proximity to the retina and relatively easy accessibility. Although intravitreous injections have long been the primary method for treating posterior segment disorders, recent successes in non-invasive topical treatments have led to the exploration of alternative administration routes. The objective of our study is to establish a platform for a Parallel Artificial Membrane Permeability Assay (PAMPA) model that mimics the posterior segment, modelling the permeation process of compounds applied topically on the eye. The study demonstrates the acceptor-phase effects of sodium hyaluronate and agar on passive permeability, while applying a previously published vitreous humor-mimetic material. Physicochemical similarities of the acceptor phase media and freshly excised porcine vitreous humor further support the applicability of the mimetic material, based on viscosity and zeta potential measurement. As a result, a new concept is introduced for measuring posterior segment permeability, with potential for future high throughput screening applications.