Xenogeneic pericardium is the basis for aortic valve (AV) bioprosthesis fabrication. State-of-the-art cross linking via glutaraldehyde (GA)-fixation is the cause for deterioration of bioprostheses within 10 to 15 years resulting in the need for re-operation of patients. Alternative cross-linking strategies such as riboflavin/UV-irradiation are therefore focus of current research. Recently, the SULEEI (sterilization and cross-linking combined with ultraviolet irradiation and low-energy electron irradiation) procedure has been introduced as innovative pericardium preparation protocol with promising in vitro and in vivo properties. Here, collagen cross-linking mechanisms underlying biomechanical stiffening of SULEEI-treated pericardium and their spatial homogeneity were analyzed using Fourier-transformed infrared (-IR) spectroscopic imaging. In parallel, ECM component quantification revealed no changes in collagen and elastin content but a reduction of glycosaminoglycan (GAG) content after SULEEI-preparation in comparison to native counterparts. No new covalent bonds were detected in UV-irradiated SULEEI pericardium. Nevertheless, variations in the respective spectral profiles were observed at 1546 and 1656 cm−1 amide II and amide I characteristic. These changes can be attributed primarily to a bridging of the amide groups and indicate that molecular forces are the basis for the increased mechanical stability by elastic moduli correlating with the wavelength shift of spectra in IR-spectroscopy. Furthermore, IR imaging demonstrated a homogeneous distribution of these intermolecular bonds and, consequently, uniform UV irradiation penetration. Therefore, a homogeneous modification of collagen is defined for stabilization of SULEEI-pericardium intended for AV replacement, as it induces polar non-covalent molecular forces that correlate with pericardium stiffening.