Abstract Intracellular Ca 2+ signals of oligodendroglia, the myelin-forming cells of the central nervous system, regulate vital cellular processes including myelination. However, studies on oligodendroglia Ca 2+ signal dynamics are still scarce, especially during myelin repair, and there are no software solutions to properly analyze the unique Ca 2+ signal characteristics in these cells. Here, we provide a comprehensive experimental and analytical workflow to acquire and analyze Ca 2+ imaging data of oligodendroglia at the population and single-cell levels in preclinical mouse models of myelin repair. We report diverse ex vivo and in vivo experimental protocols to obtain reproducible Ca 2+ imaging data from oligodendroglia in demyelinated lesions. Importantly, we provide an analytical pipeline containing two free, open source and cross-platform software programs, Occam and post-prOccam, that enable the fully automated analysis of one- and two-photon Ca 2+ imaging datasets from oligodendroglia obtained by either ex vivo or in vivo Ca 2+ imaging techniques. This versatile and accessible experimental and analytical framework, which revealed significant but uncorrelated spontaneous Ca 2+ activity in oligodendroglia inside demyelinated lesions, should facilitate the elucidation of Ca 2+ -mediated mechanisms underlying remyelination and therefore help to accelerate the development of therapeutic strategies for the many myelin-related disorders, such as multiple sclerosis.