IntroductionPolar metabolites inCaenorhabditis elegans (C. elegans)have predominantly been analyzed using hydrophilic interaction liquid chromatography coupled to mass spectrometry (HILIC-MS). Capillary electrophoresis coupled to mass spectrometry (CE-MS) represents another complementary analytical platform suitable for polar and charged analytes.ObjectiveWe compared CE-MS and HILIC-MS for the analysis of a set of 60 reference standards relevant forC. elegansand specifically investigated the strengths of CE separation. Furthermore, we employed CE-MS as a complementary analytical approach to study polar metabolites inC. eleganssamples, particularly in the context of longevity, in order to address a different part of its metabolome.MethodWe analyzed 60 reference standards as well as metabolite extracts fromC. elegans daf-2loss-of-function mutants and wild-type (WT) samples using HILIC-MS and CE-MS employing a Q-ToF-MS instrument.ResultsCE separations showed narrower peak widths and a better linearity of the estimated response function across different concentrations which is linked to less saturation of the MS signals. Additionally, CE exhibited a distinct selectivity in the separation of compounds compared to HILIC-MS, providing complementary information for the analysis of the target compounds. Analysis ofC. elegansmetabolites ofdaf-2mutants and WT samples revealed significant alterations in shared metabolites identified through HILIC-MS, as well as the presence of distinct metabolites.ConclusionCE-MS was successfully applied inC. elegansmetabolomics, being able to recover known as well as identify novel putative biomarkers of longevity.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11306-023-02025-7.