AbstractSubstantial efforts are underway to deepen our understanding of human brain morphology, structure, and function using high‐resolution imaging as well as high‐content molecular profiling technologies. The current work adds to these approaches by providing a comprehensive and quantitative protein expression map of 13 anatomically distinct brain regions covering more than 11,000 proteins. This was enabled by the optimization, characterization, and implementation of a high‐sensitivity and high‐throughput microflow liquid chromatography timsTOF tandem mass spectrometry system (LC–MS/MS) capable of analyzing more than 2,000 consecutive samples prepared from formalin‐fixed paraffin embedded (FFPE) material. Analysis of this proteomic resource highlighted brain region‐enriched protein expression patterns and functional protein classes, protein localization differences between brain regions and individual markers for specific areas. To facilitate access to and ease further mining of the data by the scientific community, all data can be explored online in a purpose‐built R Shiny app (https://brain‐region‐atlas.proteomics.ls.tum.de).