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The optimization of collagen-based scaffolds for tissue engineering goes through the careful selection of the crosslinking method(s), which should impart the prerequisite mechanical and degradation properties without impairing the cell/tissue response. Here, we investigated the chemically effective (ρxch) and the elastically effective (ρxel) crosslink density of collagen-based scaffolds, induced by various crosslinking methods. The aim was to get a deeper insight into the influence of intramolecular and intermolecular crosslinks on several scaffold properties. Freeze-dried collagen matrices were crosslinked via a dehydrothermal treatment (DHT), and then treated with different chemical agents, including carbodiimide (EDC), glutaraldehyde (GTA), formaldehyde (FA), genipin (GP) and dimethyl suberimidate (DMS). Quantification of primary amines and stress-relaxation compressive tests were performed to evaluate ρxch and ρxel, respectively. Scaffolds were then assessed for their water uptake, thermal stability and in vitro resistance to enzymatic degradation. Interestingly, for the various crosslinking treatments ρxch was found to increase in the order DHT