Histology-defined proteomics investigations are a fundamental tool in modern molecular pathology. Molecular signatures of specific cells must be detected within their correct histopathological context to draw conclusions regarding disease development and progression. The proteomics group at the Fondazione Pisana per la Scienza ONLUS is developing advanced mass spectrometry protocols for proteomics characterization of oncological and neurological samples. This involves mass spectrometry imaging (MSI), for its seamless integration with histopathological analysis, as well as histology/MSI directed laser capture microdissection for in-depth quantitative proteomics 1,2. The LC-MS/MS based proteomics on microdissected samples requires the miniaturization of the traditional bottom-up protocols.