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A better understanding of the interactions between biological entities and nanostructures is of central importance for developing functionalized materials and systems such as active surfaces with adapted biocompatibility. There is clear evidence in literature that cells and proteins generally interact with nanoscale-featured surfaces. Despite this quantity of information, little is known about the functional relationship between surface properties (i.e., roughness and nanostructuration) and biomolecules interaction. The main obstacle in the achievement of this goal is a technological one. Precise and straightforward control on surface modification at the nanometer level is required for understanding how nanostructuration influences interactions at bio/nonbio interface. In this paper, the authors describe the advantages of the focused ion beam (FIB) for surface nanostructuration of any material. The use of light transmitting substrates (especially glass) is often useful when studying the influence of surface morphology-in terms of shape and feature size-on bio/nonbio interactions by using traditional methods of biology and biotechnology. A simple methodology enabling a very efficient patterning of glass surfaces is thus described and validated: the enhancement of proteins interaction on FIB-nanostructured glass surfaces is demonstrated via fluorescence assays and a relationship between the adsorbed protein concentration and the density of surface patterning is derived.