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Summary: Here, we present a protocol to assess demyelination in the corpus callosum of an acute cuprizone mouse model, which is routinely used to induce demyelination for studying myelin regeneration in the rodent brain. We describe the tracing of neural stem cells via intraperitoneal injection of tamoxifen into adult Gli1CreERT2;Ai9 mice and the induction of demyelination with cuprizone diet. We also detail EdU administration, cryosectioning of the mouse brain, EdU labeling, and immunofluorescence staining to examine proliferation and myelination.For complete details on the use and execution of this protocol, please refer to Radecki et al. (2020).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.