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Proteomics offers vast potential for studying the molecular regulation of the human brain. Formalin fixation is a common method for preserving human tissue; however, it presents challenges for proteomic analysis. In this study, we compared the efficiency of two different protein-extraction buffers on three post-mortem, formalin-fixed human brains. Equal amounts of extracted proteins were subjected to in-gel tryptic digestion and LC-MS/MS. Protein, peptide sequence, and peptide group identifications; protein abundance; and gene ontology pathways were analyzed. Protein extraction was superior using lysis buffer containing tris(hydroxymethyl)aminomethane hydrochloride, sodium dodecyl sulfate, sodium deoxycholate, and Triton X-100 (TrisHCl, SDS, SDC, Triton X-100), which was then used for inter-regional analysis. Pre-frontal, motor, temporal, and occipital cortex tissues were analyzed by label free quantification (LFQ) proteomics, Ingenuity Pathway Analysis and PANTHERdb. Inter-regional analysis revealed differential enrichment of proteins. We found similarly activated cellular signaling pathways in different brain regions, suggesting commonalities in the molecular regulation of neuroanatomically-linked brain functions. Overall, we developed an optimized, robust, and efficient method for protein extraction from formalin-fixed human brain tissue for in-depth LFQ proteomics. We also demonstrate herein that this method is suitable for rapid and routine analysis to uncover molecular signaling pathways in the human brain.