ConspectusToll-like receptors (TLRs) are the "gatekeepers" of the immune system in humans and other animals to protect the host from invading bacteria, viruses, and other microorganisms. Since TLR4 was discovered as the receptor for endotoxin in the late 1990s, significant progress has been made in exploiting an understanding of the function of TLRs. The TLR-signaling pathway is crucial for the induction and progression of various diseases. Dysregulation of TLR signaling contributes to numerous pathological conditions, including chronic inflammation, sepsis, cancers, asthma, neuropathic pain, drug addiction, and autoimmune diseases. Therefore, manipulation of TLR signaling is promising to halt their activity in inflammatory diseases, to enhance their signaling to fight cancers, to modulate their role in autoimmune diseases, and to suppress them to treat drug addiction. TLR agonists have demonstrated great potential as antimicrobial agents and vaccine adjuvants, whereas TLR antagonists are being developed as reagents and drugs to dampen immune responses. Because of their pivotal potential therapeutic applications, fruitful small-molecule compounds and peptide fragments have been discovered, and many of them have advanced to various stages of clinical trials (though only two have been approved by the Food and Drug Administration (FDA): MPLA as a TLR4 agonist and imiquimod as a TLR7 agonist).In this Account, we focus on the progress in developing TLR signaling pathway modulators (mainly focused on the Yin and Wang laboratories) over the past decade and highlight the accomplishments and currently existing challenges in the development of TLR modulators. First, we briefly describe the members of the human TLR family along with their natural modulators. Second, we illustrate our endeavors to discover TLR-targeted agents using comprehensive approaches. Specifically, a discussion of identification and characterization of new chemical entities, determination of modes of action, and further applications is presented. For instance, the TLR3 antagonist was first discovered through screening, and the inhibitory activity was confirmed in murine cells. Considering the glycosylation on TLR3, a new direction for TLR3 modulator design was pointed out to target asparagine glycosylation. We have particularly focused on the discovery of TLR4 antagonists and have assessed their great potential in the clinical treatment of drug addiction and alcohol use disorders. In addition, we discuss multiple other popular and robust techniques for modulator discovery. Not only small organic modulators but also stapled peptides and peptidomimetics will attract more and more attention in the future. Finally, current challenges, opportunities, and future perspectives for TLR-targeted agents are also discussed.