Activation of innate immune sensors by endogenous DNA and RNA can lead to autoimmune and autoinflammatory diseases. Quantification of the unperturbed phosphoprotein content in immune cells provides insight into the spontaneous activity of immune signaling pathways triggered by nucleic acid recognition. Here, we present a phosphoflow protocol for measuring phosphoproteins in mouse models of autoimmunity that incorporates strategies to preserve native phosphoprotein levels during sample collection and to reliably detect low signaling activity common in chronic disease states. For complete details on the use and execution of this protocol, please refer to Jütte et al. (2021).