Spatial molecular profiling of complex tissues is essential to investigate cellular function in physiological and pathological states. However, methods for molecular analysis of large biological specimens imaged in 3D are lacking. Here, we present DISCO-MS, a technology that combines whole-organ/whole-organism clearing and imaging, deep-learning-based image analysis, robotic tissue extraction, and ultra-high-sensitivity mass spectrometry. DISCO-MS yielded proteome data indistinguishable from uncleared samples in both rodent and human tissues. We used DISCO-MS to investigate microglia activation along axonal tracts after brain injury and characterized early- and late-stage individual amyloid-beta plaques in a mouse model of Alzheimer's disease. DISCO-bot robotic sample extraction enabled us to study the regional heterogeneity of immune cells in intact mouse bodies and aortic plaques in a complete human heart. DISCO-MS enables unbiased proteome analysis of preclinical and clinical tissues after unbiased imaging of entire specimens in 3D, identifying diagnostic and therapeutic opportunities for complex diseases. [Display omitted] [Display omitted] • DISCO-MS is a spatial proteomics technology in optically cleared whole specimens • DISCO-MS is aided by AI and robotics and yields proteome similar to fresh samples • DISCO-bot-aided DISCO-MS reveals spatial immune cell heterogeneity in mouse bones • DISCO-bot-aided DISCO-MS reveals plaque heterogeneity in human coronary artery DISCO-MS and DISCO-bot allows for unbiased spatial proteome analysis of small tissue regions identified by panoptic imaging of large cleared samples, in silico reconstructions, and automated minimally invasive robotic tissue extraction. [ABSTRACT FROM AUTHOR]