Many viral proteins bind to DNA and play critical roles in the protection or stabilization of the viral genome during infection, capsid shell synthesis, and entire lifespan of the virus. Moreover, the interaction between a viral protein or host cell protein and its DNA can regulate virus recognition, host infection, and host–virus interactions and affect antiviral immunity. Thus, a method is needed to confirm the interaction between viral proteins and DNA, which would provide a better understanding of the mechanism and function of viral proteins and DNA and the regulation of antiviral immunity. An electrophoretic mobility shift assay (EMSA) with a biotin-labeled probe provides a simple way to determine the interaction between viral proteins and DNA during the virus lifecycle and antiviral immunity. This chapter presents detailed information and experimental procedures for measuring the interaction between target proteins and DNA via EMSA.