SummaryTumor relapse is the leading adverse prognostic factor in medulloblastoma (MB). However, there is still no established mouse model for MB relapse, impeding our efforts to develop strategies to treat relapsed MB. We present a protocol for generating a mouse model for relapsed MB using irradiation by optimizing mouse breeding and age, as well as irradiation dosage and timing. We then detail procedures for determining tumor relapse based on tumor cell trans-differentiation in MB tissue, immunohistochemistry, and tumor cell isolation.For complete details on the use and execution of this protocol, please refer to Guo et al. (2021).1