Picornaviridae is a large family of single-stranded positive RNA viruses that can infect both humans and animals. These include the enteroviruses (e.g. poliovirus, coxsackievirus and rhinoviruses) as well as the cardioviruses (e.g. encephalomyocarditis virus (EMCV)). Picornaviruses have evolved to interact, use and/or evade cellular host systems to create the optimal environment for viral growth and spreading. It is known that these viruses can modify kinase activity during infection, but a comprehensive overview of the (de)regulation of cellular kinases during picornavirus infection is lacking. To study the landscape of kinase activity during picornavirus infection, we here applied dedicated targeted mass spectrometry-based assays targeting ~40% of the human kinome. Our data show that kinases of the MAPK pathways were activated (e.g. ERK1/2, RSK1/2, JNK1/2/3, p38), while kinase involved in regulating the cell cycle (e.g. CDK1/2, GWL, DYRK3) were inactivated. Additionally, we observed the activation of CHK2, an important kinase of the DNA damage response. Using pharmacological kinases inhibitors, we demonstrate that some of the activated kinases are essential for the replication of EMCV. Altogether, we provide a quantitative system-wide understanding of the regulation of kinome activity induced by picornavirus infection, which is important for developing novel therapeutic interventions.