<jats:p> Small molecule metabolites play important roles in <jats:italic>Caenorhabditis elegans</jats:italic> biology, but effective approaches for identifying their chemical structures are lacking. Recent studies revealed that a family of glycosides, the ascarosides, differentially regulate <jats:italic>C. elegans</jats:italic> development and behavior. Low concentrations of ascarosides attract males and thus appear to be part of the <jats:italic>C. elegans</jats:italic> sex pheromone, whereas higher concentrations induce developmental arrest at the dauer stage, an alternative, nonaging larval stage. The ascarosides act synergistically, which presented challenges for their identification via traditional activity-guided fractionation. As a result the chemical characterization of the dauer and male attracting pheromones remained incomplete. Here, we describe the identification of several additional pheromone components by using a recently developed NMR-spectroscopic approach, differential analysis by 2D NMR spectroscopy (DANS), which simplifies linking small molecule metabolites with their biological function. DANS-based comparison of wild-type <jats:italic>C. elegans</jats:italic> and a signaling-deficient mutant, <jats:italic>daf-22</jats:italic> , enabled identification of 3 known and 4 previously undescribed ascarosides, including a compound that features a <jats:italic>p</jats:italic> -aminobenzoic acid subunit. Biological testing of synthetic samples of these compounds revealed additional evidence for synergy and provided insights into structure–activity relationships. Using a combination of the three most active ascarosides allowed full reconstitution of the male-attracting activity of wild-type pheromone extract. Our results highlight the efficacy of DANS as a method for identifying small-molecule metabolites and placing them within a specific genetic context. This study further supports the hypothesis that ascarosides represent a structurally diverse set of nematode signaling molecules regulating major life history traits.