Chemical communication forms an integral part of any organism’s overall health, longevity, social interactions and various other aspects of its biology. Chemical cues produced by different organisms are products of primary and secondary metabolism, and are highly specific in their structural assembly and signaling functions. Minute differences in structures and functions of these metabolites can, for example, help an organism distinguish between pathogenic and beneficial microbes, cause the organism to extend or reduce its lifespan, signal availability or lack of sustenance, and provide snapshots of its metabolic state. As such, it is crucial to annotate structures and functions of the underlying chemical causes behind biological effects, understand how subtle structural differences cause dramatic changes in observed phenotypes, and elucidate their biosynthetic mechanisms to learn how biological information is encoded in these signaling molecules. Ultra-high-performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) is rapidly becoming an indispensable tool in the study of signaling molecules using comparative metabolomics. This technique far outdoes classical activity-guided fractionation methods to identify chemical cues in biological systems in terms of required time, ease of use and sensitivity. The use of both activity-guided fractionation and UHPLC-MS/MS techniques to elucidate structures of signaling molecules involved in host-pathogen interactions between nematodes and bacteria, and mitochondrion-mediated longevity in nematode are discussed in this dissertation. Forward genetic approaches to identify biosynthetic enzymes responsible for the highly specific assembly of nematode-derived modular metabolites, and validation of their biosynthetic functions are also described. Finally, an untargeted metabolomics approach combining high resolution UHPLC-MS/MS data with genome-wide association studies to annotate the nematode metabolome and identify candidate biosynthetic genes for various metabolites is evaluated.