Understanding the interaction mechanisms of phospholipids with surfaces is crucial for the exploitation of lipid bilayers as models of the cell membrane as well as templates for biosensors. Moreover, controlling and manipulating lipid nanoparticles for the investigation of their properties by means of single-particle sensitive surface techniques require the ability to tailor the chemical properties of surfaces to achieve a stable and sparse binding of lipid particles, while keeping them from aggregating, or denaturing. Here we present a quantitative morphological and structural investigation by atomic force microscopy of supported phospholipid layers and nanostructures on cholesterol-functionalized glass surfaces, in comparison with other surfaces with different interfacial properties. We show that the functionalization of glass coverslips with cholesterol groups is a viable route for the production of optically transparent, scanning probe microscopy-compatible clean substrates for the effective immobilization of both extended single lipid bilayers and lipid nanoparticles.