Myelin, the multilayered membrane that enwraps and insulates neuronal axons for fast signal propagation, is a plasma membrane specialization of oligodendrocytes and Schwann cells in the central and peripheral nervous system, respectively. Here we provide our lab protocols for the purification of myelin from mouse brains and for gel-based and gel-free proteomic applications, as well as a brief discussion with respect to our current knowledge of the myelin cytoskeleton.