Basement membranes (BMs) are sheets of extracellular matrix that separate epithelia from connective tissues and outline muscle fibers and the endothelial lining of blood vessels. A major function of basement membranes is to establish and maintain stable tissue borders. We introduce the inner limiting membrane (ILM), located at the retinal-vitreal junction, as a model system for studying the biophysical properties of BM. We also introduced atomic force microscopy techniques as important tools to investigate the ILM under physiologically relevant conditions. We were able to determine changes in both the thickness and elasticity of chick ILM during embryonic development. We also determined that BMs are much thicker in their native state than previously thought. Proteoglycans, specifically their heparan sulfate side-chains, were found to significantly contribute to ILM biophysical properties. The effects of aging on the composition, structure, and biophysical properties of the adult human ILM were investigated. A compositional shift in the ILM was associated with an increase in both the ILM thickness and elastic modulus during aging. The role of heparan sulfate molecules in the human ILM was determined to be similar to that of the chick ILM. The biophysical changes in mouse knockout models for congenital muscular dystrophy were also investigated. These models had protein knockouts that inhibit the proper formation of basement membranes. There were differences in the biophysical properties along with visually noted disruptions in the mouse inner limiting membranes due to improper formation of the BM. This study provided novel insight into the biophysical importance of BMs and the ability to study changes associated with a variety of biological conditions that are relevant to proper biological function, in addition to setting a baseline for the biophysical properties of BMs.