AbstractMetabolomics has grown into one of the major approaches for systems biology studies, in part driven by developments in mass spectrometry (MS), providing high sensitivity and coverage of the metabolome at high throughput. Untargeted metabolomics allows for the investigation of metabolic phenotypes involving several hundreds to thousands of metabolites. In this approach, all signals in a mass chromatogram are processed in an unbiased way, allowing for a deeper investigation of metabolic phenotypes, but also resulting in significantly more complex data processing and post‐processing steps. In this article, we discuss all the intricacies involved in extracting and analyzing metabolites by chromatography coupled to MS, as well as the processing and analysis of such datasets. © 2019 The Authors.Basic Protocol 1: Metabolite extraction for LC‐MSAlternate Protocol: Methyl tert‐butyl ether (MTBE) extraction for multiple mass spectrometry platforms (GC‐polar, LC‐polar, LC‐lipid)Basic Protocol 2: LC‐MS analysisSupport Protocol 1: GC‐MS derivatization and analysisSupport Protocol 2: Lipid analysisBasic Protocol 3: LC‐MS data processingBasic Protocol 4: Data analysisBasic Protocol 5: Metabolite annotationSupport Protocol 3: Molecular networking using MetNetSupport Protocol 4: Co‐injection of authentic standards