Oligodendrocytes (OLs) are glial cells that myelinate axons in the central nervous system (CNS). These cells are generated from oligodendrocyte precursor cells (OPCs) during CNS development, and the population of OPCs that remain in the adult is responsible for spontaneous remyelination in demyelinating diseases such as multiple sclerosis (MS). Thus, OPCs represent an interesting source for regenerative therapies. Here, we describe an updated protocol for isolation of OPCs based on the magnetic cell separation (MACS) technology and oligodendroglial lineage-specific markers. After the extraction of nerve tissue, mechanical and enzymatic dissociation is performed. The cells are then immunomagnetically labeled and separated through columns composed of a matrix of ferromagnetic spheres coupled to the magnetic field of the MACS separator. This method has several advantages: it is significantly more efficient and faster if compared to other conventional methods, such as flow cytometry or shaking. In addition, the OPCs isolated in this way are significantly more viable, which facilitates their use of these cells in different experimental approaches, both in vitro and in vivo.