We report the study of the morphology, topography, and adhesion properties of internal limiting membrane (ILM) from patients with macular holes. The quantitative analysis of human ILM could provide essential information toward the improvement of existing surgical instruments for more efficient and safer surgical removal of ILM. Imaging in air revealed the presence of globular structures in most of the samples analyzed which were coupled with fibrillar structures in some of the samples. Modification of silicon nitride AFM tips with oppositely charged functional groups showed changes in adhesion force at the membrane-tip interface. Defining the surface characteristics of the human ILM is an initial step in the development of improved surgical tools that may allow nontraumatic stripping of ILM during surgery.