The adsorption behavior of collagen on solid surfaces is a process that determines the role of this protein to mediate cell–material interaction. Herein, the mechanism of self‐assembly and organization of collagen on a model substrate is investigated in the presence of TiO2 nanoparticles. In solution, results show that nanoparticles do not alter the conformation of collagen (triple‐helix), and slightly delay the kinetics of its self‐assembly. In the adsorbed state, by exploring the dewetting patterns of collagen layers from atomic force microscopy (AFM) images, a method is developed to extract parameters describing the characteristics of collagen networks. It is shown that collagen layer is strongly impacted by the presence of nanoparticles in the medium. These results are consistent with the analysis of the protein layers in the hydrated state, showing a rigidification, as observed by quartz crystal microbalance with dissipation monitoring (QCM‐D), and the formation of shorter and/or less extended fibrillar structures with a lower surface density, as probed by AFM force spectroscopy. The approach described here provides a reconciliation between disparate views of collagen layers' characterization in the dried and the hydrated states. It also offers new perspectives to assess the impact of nanoparticles on the organization of collagen during in vitro tests, particularly at the stage of cell adhesion.