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The analysis of brain function in normal aging and neurodegenerative, psychiatric, and neurological diseases has long been a subject of interest and has historically been investigated through descriptive analysis of macroscopic or microscopic observations. It is now possible to characterize brain cells, such as neurons and glial cells, or even their subcellular components, at the molecular level. This ability enables researchers to more closely examine brain cell specific molecular pathways to elucidate distinct brain functions. Furthermore, the analysis of neuronal maintenance and disease-causing effects is a central component of neurological investigations, which include proteomic approaches. Proteomics allows the identification of thousands of proteins through descriptive and comparative analyses and can provide a detailed overview of a distinct cellular state. Such analyses often require the isolation of individual cell types or subcellular components to investigate specific questions. This review provides an overview of the currently applied state-of-the-art prefractionation strategies in this field.