The degeneration of heart valve bioprostheses due to calcification processes is caused by the intercalation of calciumhydroxyapatite in pericardium collagen bundles. Variations of the protein secondary structure of biomaterials according to preparation are relevant for this mineralization process and thus the structural characterization of innovative bioprostheses materials is of great importance. The gold standard for prostheses preparation is glutaraldehyde (GA)-fixation of bovine pericardium that adversely promotes calcification. The novel GA-free SULEEI-treatment of bovine pericardium includes decellularization, UV-crosslinking, and electron beam sterilization. The aim of this study is the structural characterization of SULEEI-treated and GA-fixed bovine pericardium. IR spectroscopic imaging combined with multivariate data and curve fit analysis was applied to investigate the amide I and amide II regions of SULEEI-treated and GA-fixed samples. The spectroscopic images of GA-fixed pericardial tissue exhibited a generally high content of amine groups and side chains providing nucleation points for calcification processes. In contrast, in SULEEI-treated tissue, the typical α-helical structure was retained and was supposed to be less prone to deterioration.