A new experimental methodology combining concepts of classical mechanical testing with basic microfabrication techniques has been developed to investigate the mechanical architecture of cytoskeletal networks and anchoring junctions within a monolayer of normal human epidermal keratinocytes (NHEKs) before, during, and after the in vitro formation of an epithelial sheet. Referred to as composite diaphragm inflation (CDI), the method resembles a microscale model of a classical axisvmmetric biaxial membrane inflation experiment. CDI utilizes a free-standing polydimethylsiloxane (PDMS) elastomer membrane as a substrate for the culture of NHEKs into an epithelial sheet via calcium induction of cytoskeletal network reorganization and anchoring junction assembly. Together, the NHEK monolayer and the PDMS membrane form a composite diaphragm suitable for mechanical measurements. If the geometry and elastic properties of the PDMS membrane are scaled appropriately, then the prestress and apparent stiffness of the NHEK layer can be characterized by measured deviations in the load-displacement behavior of the composite diaphragm with respect to the behavior of the PDMS membrane measured prior to cell culture.