Proteomics studies employing primary neurons are difficult due to the neurons' characteristics. We have developed a detergent-based fractionation method which reduces complexity of the protein extracts, is sufficiently fast to allow differential proteomics analysis after treatments of neurons for short time periods, can be applied to small numbers of cells directly in culture plates, and allows differential extraction of proteins in a compartment-specific manner. The sequential use of detergent-containing buffers on neurons in culture plates yields four extracts enriched in cytosolic, membrane-bound or enclosed, nuclear, and cytoskeletal proteins. Fractionation of neurons was validated by comparison of the distribution of known subcellular marker proteins in the four extracts using Western blotting. Comparison of extracts by DIGE showed a clear difference in protein composition demonstrating significant variations with a fold change (FC) of at least 1.20 for 82% of the detected spots. Using proteins identified in these spots that could be assigned a subcellular localization based on descriptions in the Uniprot database, an extraction efficiency of 85% was calculated for cytosolic proteins in extract 1, 90% for membrane-bound and membrane-enclosed proteins in extract 2, 82% for nuclear proteins in extract 3 and 38% for cytoskeletal and RAFT proteins in extract 4.