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Invasion of pathogenic microorganisms or tissue damage activates innate immune signaling receptors that sample subcellular locations for foreign molecular structures, altered host molecules, or signs of compartment breaches. Upon engagement of innate immune receptors an acute but transient inflammatory response is initiated, aimed at the clearance of pathogens and cellular debris. Among the molecules that are sensed are nucleic acids, which activate several members of the transmembrane Toll‐like receptor (TLR) family. Inappropriate recognition of nucleic acids by TLRs can cause inflammatory pathologies and autoimmunity. Here, we review the mechanisms involved in triggering nucleic acid‐sensing TLRs and indicate checkpoints that restrict their activation to endolysosomal compartments. These mechanisms are crucial to sample the content of endosomes for nucleic acids in the context of infection or …