The desired mechanical and biological performances of collagen that have led to its broad application as a building block in the biomedical field attributed to its intrinsic hierarchical structure from the nanoscale to macroscale, are discussed herein. Modulating the self-assembly process using regulatory factors can lead to obtaining collagenous materials with tuneable functional performance, which can then determine distinctive cellular responses. Herein, we present an overview of the corresponding characterization techniques used to detect the changes in light transmittance, architecture and mechanics during collagen fibrillogenesis. By combining regulatory parameters with characterization methods, researchers can selectively fabricate collagenous biomaterials with various functional responses.