Purpose: Histopathological evaluation presents conflicting reports regarding aortic abnormalities. The authors aim to present proof-of-concept study to explore the feasibility of matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) in combination with histopathology for characterizing alterations in the aneurysmal ascending formalin-fixed paraffin-embedded (FFPE) aorta tissue. Experimental design: The authors assess FFPE specimens from patients with a dilated aorta and bicuspid aortic valve (BAV), those with a standard tricuspid aortic valve (TAV), and those with Marfan syndrome (MFS) via histopathology and grade the conditions for elastic fiber fragmentation (EFF) and MALDI-IMS. The proteins using liquid chromatographic–mass spectrometry are identified and the results are confirmed by immunohistochemistry. Results: There is significant difference in terms of EFF between MFS and BAV, and TAV and BAV. Characteristic peptide signatures and m/z values in the EFF facilitate the characterization among the aortic specimens of BAV, MFS, and TAV. The m/z values from the aortic alpha smooth muscle actin and myosin heavy chains significantly increase in BAV compared with MFS and TAV. These findings are confirmed by immunohistochemistry. Conclusion: The results represent a strategy that uses MALDI-IMS in combination with histopathology as promising approaches to characterize spatial alteration in the structure of the aneurysmal ascending aorta.