Glial cells are important players in the pathogenesis of multiple sclerosis (MS)-induced injury to the central nervous system. Characterization of their roles is a crucial milestone to advance therapeutic strategies for MS patients. The focus of this chapter is to provide an overview of the methods used to study glial cells in preclinical models of MS that can be translated into the field of human MS. Firstly, microanatomy and physiology of glial cells in the healthy brain are introduced. Then, main immunohistological, electrophysiological, structural, and molecular imaging techniques to study glial cells are described. Additionally, the function of each glial cell type in the development and restoration of MS-induced damage is highlighted. Finally, evidence from preclinical studies on glial cells as potential targets to be approached in order to design novel therapies for MS patients is discussed.