SUMMARYSpatial molecular profiling of complex tissues is essential to investigate cellular function in physiological and pathological states. However, methods for molecular analysis of biological specimens imaged in 3D as a whole are lacking. Here, we present DISCO-MS, a technology combining whole-organ imaging, deep learning-based image analysis, and ultra-high sensitivity mass spectrometry. DISCO-MS yielded qualitative and quantitative proteomics data indistinguishable from uncleared samples in both rodent and human tissues. Using DISCO-MS, we investigated microglia activation locally along axonal tracts after brain injury and revealed known and novel biomarkers. Furthermore, we identified initial individual amyloid-beta plaques in the brains of a young familial Alzheimer’s disease mouse model, characterized the core proteome of these aggregates, and highlighted their compositional heterogeneity. Thus, DISCO-MS enables quantitative, unbiased proteome analysis of target tissues following unbiased imaging of entire organs, providing new diagnostic and therapeutic opportunities for complex diseases, including neurodegeneration.Graphical AbstractHighlightsDISCO-MS combines tissue clearing, whole-organ imaging, deep learning-based image analysis, and ultra-high sensitivity mass spectrometryDISCO-MS yielded qualitative and quantitative proteomics data indistinguishable from fresh tissuesDISCO-MS enables identification of rare pathological regions & their subsequent molecular analysisDISCO-MS revealed core proteome of plaques in 6 weeks old Alzheimer‘s disease mouse model Supplementary Video can be seen at: http://discotechnologies.org/DISCO-MS/