Severe aortic stenosis (AS) is prevalent in adults ≥ 65 years, a significant cause of morbidity and mortality, with no medical therapy. Lipid and proteomic alterations of human AS tissue were determined using mass spectrometry imaging (MSI) and liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) to understand histopathology, potential biomarkers of disease, and progression from non-calcified to calcified phenotype. A reproducible MSI method was developed using healthy murine aortic valves (n = 3) and subsequently applied to human AS (n = 2). Relative lipid levels were spatially mapped and associated with different microdomains. Proteomics for non-calcified and calcified microdomains were performed to ascertain differences in expression. Increased pro-osteogenic and inflammatory lysophosphatidylcholine (LPC) 16:0 and 18:0 were co-localized with calcified microdomains. Proteomics analysis identified differential patterns in calcified microdomains with high LPC and low cholesterol as compared to non-calcified microdomains with low LPC and high cholesterol. Calcified microdomains had higher levels of: apolipoproteins (Apo) B-100 (p < 0.001) and Apo A-IV (p < 0.001), complement C3 and C4-B (p < 0.001), C5 (p = 0.007), C8 beta chain (p = 0.013) and C9 (p = 0.010), antithrombotic proteins alpha-2-macroglobulin (p < 0.0001) and antithrombin III (p = 0.002), and higher anti-calcific fetuin-A (p = 0.02), while the osteoblast differentiating factor transgelin (p < 0.0001), extracellular matrix proteins versican, prolargin, and lumican ( p < 0.001) and regulator protein complement factor H (p < 0.001) were higher in non-calcified microdomains. A combined lipidomic and proteomic approach provided insight into factors potentially contributing to progression from non-calcified to calcific disease in severe AS. Additional studies of these candidates and protein networks could yield new targets for slowing progression of AS.