C-type lectin receptors (CLRs) play pivotal roles in recognizing specific structures of carbohydrate-based ligands and in regulating innate immune responses. Several CLRs are known to recognize glycolipid ligands derived from microbial or endogenous sources. Among these glycolipid-recognizing receptors, this review focuses on Mincle and DCAR, summarizing the structural features and immunological activities of representative or recently developed synthetic ligands, with particular emphasis on their application to the design of functional molecular probes. These ligand-based probes have proven useful not only for elucidating receptor–ligand interactions, but also for visualizing the dynamics of the receptors in live cells. Notably, we have established fluorescence-labeled molecular probes derived from synthetic glycolipids, which have enabled the analysis of intracellular trafficking of both Mincle and its ligands. Such molecular tools offer valuable insights into the mechanisms of CLR-mediated immune signaling and contribute to the development of innovative therapeutic and diagnostic strategies.