SummaryMyoblast determination protein 1 (MyoD) dynamics define the activation status of muscle stem cells (MuSCs), aiding in muscle tissue regeneration after injury. However, the lack of experimental platforms to monitor MyoD dynamicsin vitroandin vivohas hampered the investigation of fate determination and heterogeneity of MuSCs. Herein, we report a MyoD knock-in (MyoD-KI) reporter mouse expressing tdTomato at the endogenousMyoDlocus. Expression of tdTomato in MyoD-KI mice recapitulated the endogenous MyoD expression dynamicsin vitroand during the early phase of regenerationin vivo. Additionally, we showed that tdTomato fluorescence intensity defines MuSC activation status without immunostaining. Based on these features, we developed a high-throughput screening system to assess the effects of drugs on the behavior of MuSCsin vitro. Thus, MyoD-KI mice are an invaluable resource for studying the dynamics of MuSCs, including their fate decisions and heterogeneity, and for drug screening in stem cell therapy.