This manuscript describes a novel method for the biofunctionalization of glass surfaces with polyhistidine-tagged proteins. The main innovation of this methodology consists of the covalent binding between the nitrilotriacetic acid (NTA) moiety and the proteins, ensuring not only orientation, but also stability of the recombinant proteins on NTA-covered surfaces. In this work, as C-terminal polyhistidine tagged cadherin extracellular fragments have been used, this methodology guarantees the proper orientation of these proteins, by mimicking their insertion into cell plasma membranes. These biofunctionalized surfaces have been characterized by confocal microscopy, X-ray photoelectron spectroscopy, contact angle, and atomic force microscopy, showing a high density of cadherins on the glass surfaces and the stability of the linkage. The prepared materials exhibited a high tendency to promote cell spreading, demonstrating the functionality of the protein and the high utility of these biomaterials to promote cell adhesion events. Interestingly, differences in the cytoskeleton organization have been observed in cells adhering to surfaces with no cadherins or with nonoriented cadherins, in comparison to surfaces functionalized with well-oriented cadherins. This method, which allows the robust immobilization of polyhistidine tagged proteins due to their covalent binding and with a defined orientation, may also find particular usefulness in the making of protein biochips, for analysis of protein-protein interactions, as well as structural and single-molecule studies.