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© 2020, Hassan et al.Mammalian cells typically start the cell-cycle entry program by activating cyclin-dependent protein kinase 4/6 (CDK4/6). CDK4/6 activity is clinically relevant as mutations, deletions, and amplifications that increase CDK4/6 activity contribute to the progression of many cancers. However, when CDK4/6 is activated relative to CDK2 remained incompletely understood. Here we developed a reporter system to simultaneously monitor CDK4/6 and CDK2 activities in single cells and found that CDK4/6 activity increases rapidly be