Our aim was to establish an effective method for protein extraction from freshly frozen human peripheral nerves, determine the minimum amount required for consistent protein extraction outcomes, and assess which method produced the highest number of protein identities. Five extraction methods were compared using 8 M urea and Ripa buffer using either the Bullet Blender or Bioruptor. Out of the total 2619 identified proteins, protein extraction using the Ripa buffer combined with either Bioruptor or Bullet Blender resulted in the identification of 1582 (60%) and 1615 (62%) proteins, respectively. In contrast, using 8 M urea and Bioruptor for protein extraction resulted in 1022 proteins (39%), whereas employing Bullet Blender yielded 1446 proteins (55%). Sample amounts, ranging from 0.6 to 10 mg, were prepared with consistent protein extraction outcome obtained for samples ≥1.2 mg. Combining Ripa and 8 M urea with Bullet Blender increased protein identification to 2126 (81%). Proteins were classified by their cell components, molecular functions, and biological processes. Furthermore, a subclassification of proteins involved in the extracellular matrix (ECM) was introduced. We recommend the use of Ripa buffer, in combination with 8 M urea and Bullet Blender for extracting proteins from fresh-frozen human nerves weighing ≥1.2 mg.