Mass spectrometry-based techniques have emerged as a gamechanger for the analysis of carbohydrates. Here, we summarize state-of-the-art glycan analysis in vacuo, demonstrating how mass spectrometry, tandem mass spectrometry, ion mobility spectrometry, and gas-phase infrared spectroscopy, supported by chromatography methods and computational modelling, facilitate the structural characterization of isolated glycans and the analysis of the entire glycome. After an introduction to glycans and techniques, the sugar classes of N-glycans, O-glycans, human milk oligosaccharides, and glycosaminoglycans will be discussed in detail, before the chapter ends with an outlook on the future of mass spectrometry-based glycan analysis.