Metabolism is the chemical transformation of small molecules in living organisms. Biogenic small molecules (BSMs) are products of metabolism that serve diverse and essential roles in biological systems. While previous research resulted in the impressive identification of the structures and biological functions of thousands of BSMs, the majority of BSMs remain uncharacterized. Three questions remain largely unanswered: (i) What are the downstream products of one specific BSM of interest, (ii) What are the metabolic intermediates, and (iii) What is the biological relevance of any one metabolite. In this work, the author developed novel approaches to address the first two questions mentioned above and characterized metabolites that are related to reduced insulin/Insulin-like growth factor signaling (IIS). Chapter 1 focuses on the incorporation of stable isotopic labels to trace the metabolism of nematode signaling molecules in plants, microorganisms, mammals, and Caenorhabditis elegans. Here, the author studied the metabolism of ascarosides, which are signaling molecules produced by nematodes that play a central role in regulating their behavior, development, and lifespan. In this chapter, we combined stable-isotope labeling and mass spectrometry-based comparative metabolomics to trace the downstream metabolites of ascarosides in various organisms and showed that ascarosides are taken up from the environment and metabolized by a wide range of phyla, including plants, fungi, bacteria, and mammals, as well as nematodes. We also showed that certain metabolic pathways are highly stereospecific. Chapters 2-3 focus on developing probes to capture biosynthetic intermediates, such as coenzyme A esters, which are reactive electrophilic species that are difficult to detect with conventional strategies. Here, we introduced hydroxylamine-based probes to convert reactive electrophilic intermediates into stable derivatives that are easily detectable via liquid chromatography-mass spectrometry (LC-MS). In Chapter 2, we used simple hydroxylamine to capture electrophilic biosynthetic intermediates. Parallel treatment with 14NH2OH and its isotopically labeled analogue 15NH2OH revealed labeled metabolites in C. elegans, Aspergillus fumigatus, and a human cell line. We also probed changes in the production of electrophilic intermediates in different biological backgrounds, e.g. C. elegans acox-1.1 mutants. However, abundances of CoA thioesters and other electrophilic intermediates are generally low, posing significant challenges for detection of their resulting hydroxylamine derivatives. Hence, in Chapter 3, we synthesized derivatives of hydroxylamine that feature either a click chemistry-capable moiety to enable enrichment or a constitutively charged ionization tag to enhance sensitivity. We found that O-(trimethylammoniobutyl)hydroxylamine (TAMOHA) dramatically enhanced the sensitivity for the detection of electrophilic intermediates, by 100-fold in the model organism C. elegans, whereas the use of click chemistry-based enrichment was not compatible with hydroxylamine-based probes. We demonstrated TAMOHA labeling, in parallel with D4-labeled homologue of TAMOHA, in wildtype C. elegans as well as to two acyl-CoA synthase mutants. Together, TAMOHA provided in-depth profiling of electrophilic intermediates and enabled to monitor their changes in different biological context. Chapter 4 focused on the metabolic signature of reduced insulin/IGF-1 signaling (IIS). IIS is a genetic pathway highly conserved in the animal kingdom that regulates metabolism, development, and aging. Reduced IIS promotes longevity in many animal model systems, including C. elegans, Drosophila, and mice. Using C. elegans as a tractable model, we employed untargeted comparative metabolomics to deeply profile IIS dependent-metabolites using three different mutants of the insulin/IGF-1 receptor daf-2 that feature mutations in different domains of the receptor and exhibit distinct longevity phenotypes. We found that the metabolome of daf-2 mutants was dramatically altered compared to wildtype. We detected changes in the metabolism of diverse lipids, amino acid catabolites, nucleosides, and ascarosides, as well as several previously uncharacterized metabolite families. We further showed that some of these metabolic changes were specifically associated with either mutations of the ligand binding domain or the receptor kinase domain. Together, we provided a blueprint for the discovery of IIS-dependent metabolites and would serve as a foundation for how IIS-dependent alteration of metabolism relates to IIS-dependent physiological changes.