The influence of hydrophobicity of the substrate surface on structural changes during protein adsorption was investigated. Plasma fibrinogen was chosen to model this effect as it is the most important protein in the body that adsorbs to foreign surfaces. Only conformations of adsorbed fibrinogen similar to that of the protein in solution do not activate the process of blood coagulation. Small spots on the substrate surface with conformational changes within the adsorbed protein are already sufficient to deteriorate biocompatibility. Mid-infrared hyperspectral imaging permits the identification of coagulated spots down to a few micrometers in size. The spectra of the FT-IR images that were assessed to be of suitable quality were clustered by a fuzzy c-means algorithm. The determination of the appropriate number of clusters was based on cluster variance. Subsequent evaluation of the centroid spectra of each cluster showed that their amide I band was separated into contributions from different structural units, with the alpha-helix content always being dominant. Significant differences between hydrophobic and hydrophilic surfaces were observed for turn and sheet contributions. Lower sheet/turn ratios appear to indicate inferior biocompatibility. Spots on hydrophilic surfaces could be identified, which exhibit structural changes similar to those on hydrophobic surfaces.