We present the use of hydrophobic paper as an effective semipermeable membrane, ideal for air-liquid interface cell culture applications. The surface properties of the paper is modified through selective CO2 laser-assisted scrubbing to create a unique porous substrate with hydrophilic regions that regulates fluid diffusion and cell attachment. To select the appropriate model, four promising hydrophobic films were compared with each other in terms of gas permeability and long-term strength in an aqueous environment (wet-strength). Among the investigated substrates, parchment paper showed the fastest rate of oxygen diffusion. Mechanical analysis revealed superior dry and wet tensile strength of 69.5 MPa and 29 MPa for parchment paper which remained virtually unchanged after 7 days of submersion in PBS. The final platform with a constant flow of media in the basal lumen was found to show confluent coverage of Calu-3 (airway epithelial cells) with a mature ZO1 expression at the air-liquid interface after 7 days of cell culture.