ABSTRACTUntargeted metabolomics via high-resolution mass spectrometry (HRMS) can reveal more than 100,000 molecular features in a single sample, many of which may represent unidentified metabolites, posing significant challenges to data analysis. We here introduce Metaboseek, an open-source analysis platform designed for untargeted comparative metabolomics and demonstrate its utility by uncovering biosynthetic functions of a conserved fat metabolism pathway, α-oxidation, usingC. elegansas a model. Metaboseek integrates modules for molecular feature detection, statistics, molecular formula prediction, and fragmentation (MS/MS) analysis, which uncovered more than 200 previously uncharacterized α-oxidation-dependent metabolites in an untargeted comparison of wildtype and α-oxidation-defectivehacl-1mutants. The identified metabolites support the predicted enzymatic function of HACL-1 and revealed that α-oxidation participates in metabolism of endogenous β-methyl-branched fatty acids and food-derived cyclopropane lipids. Our results showcase compound discovery and feature annotation at scale via untargeted comparative metabolomics applied to a conserved primary metabolic pathway and suggest a model for the metabolism of cyclopropane lipids.