A comprehensive analysis of plasma membrane proteins is essential to in-depth understanding of brain development, function, and diseases. Proteomics offers the potential to perform such an analysis, yet it requires efficient protocols for the purification of plasma membranes. Here, we present a novel and efficient protocol for the separation and enrichment of brain plasma membranes. It lasts only 4 hours and is easy to perform. It highly enriches plasma membranes and can be applied to small amounts of brain tissue, such as the cerebellum of a single rat, which has been used in the present study. The protocol is based on affinity partitioning of microsomes in an aqueous two-phase system. Marker enzyme assays demonstrated a more than 12-fold enrichment of plasma membranes and a strong reduction of other compartments, such as mitochondria and the endoplasmic reticulum. 506 different proteins were identified when the enriched proteins underwent LC-MS/MS analysis subsequent to protein separation by SDS-PAGE. Using gene ontology, 146 proteins were assigned to a subcellular compartment. Ninety-three of those (64%) were membrane proteins and 49 (34%) were plasma membrane proteins. A combined literature and database search for all 506 identified proteins revealed subcellular information on 472 proteins, of which 197 (42%) were plasma membrane proteins. These comprised numerous transporters, channels, and neurotransmitter receptors, e.g. the inward rectifying potassium channel Kir7.1 and the cerebellum-specific GABA receptor GABRA6. Surface proteins involved in cell-cell contact and disease-related proteins were also identified. Six of the 146 assigned proteins were derived from mitochondrial membranes and 5 were derived from membranes of the endoplasmic reticulum. Taken together, our protocol represents a simple, rapid, and reproducible tool for the proteomic characterization of brain plasma membranes. As it conserves membrane structure and protein interactions, it is also suitable to enrich protein complexes from plasma membranes for subsequent analysis.