Membrane transport in diffusion cell studies is not one-dimensional from the donor to the receptor. Lateral diffusion within the membrane into the surrounding clamped region can lead to edge effect. Lateral diffusion can also affect the impact of an object blocking the membrane in a diffusion cell. The effects of lateral transport on permeation across a two-layer membrane in diffusion cells were investigated in this study under edge effect and membrane blocking conditions that could be encountered in previous gingiva and hypothetical skin permeation studies. Model simulations of time-dependent and steady-state transport were performed using COMSOL Multiphysics. The simulations indicated edge effect could increase the steady-state flux across the membrane up to 35% with a relatively thick membrane and small diffusion cell opening (e.g., gingiva study). The edge effect decreased when the relative thickness and permeability of the major barrier (top layer in the two-layer membrane) decreased. When the membrane was partially blocked by an object, lateral diffusion within the membrane could mitigate its impact: e.g., when the object was in the receptor, the impact caused by membrane blocking was reduced more than half. Therefore, membrane lateral transport should be considered under certain circumstances in permeation studies using diffusion cells.