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Summary: Innate immunity is the first layer of defense against infection in mammals and is tightly regulated. We monitored cGAS/STING signaling upon ISD90 or 2′,3′-cGAMP stimulation in EA.hy926 cells by western blotting, RT-PCR, and ELISA analyses to reveal signaling activation and IFNβ production. In addition, we also include an HSV-1 infected mouse model to further reveal procedures in analyzing cGAS/STING signaling in mice. This protocol could be applied to studies focusing on cell culture or mouse models to investigate cGAS/STING signaling.For complete details on the use and execution of this protocol, please refer to Zhang et al. (2020).