Updated project metadata. Proteomics offers vast potential to study the molecular regulation of the human brain. Formalin fixation is a common method for preserving human tissue, however, 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 liquid chromatography-tandem mass spectrometry (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 tissue was analyzed by label free quantification (LFQ) proteomics, Ingenuity Pathway Analysis and PANTHERdb. Inter-regional analysis of the human brain revealed differential enrichment of proteins. We found similarly activated cellular signaling pathways, 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 further demonstrate that this method is suitable for quick analysis of molecular signaling pathways in the human brain.