Lipooligosaccharides (LOS) are powerful Gram‐negative glycolipids that evade the immune system and invade host animal and vegetal cells. The structural elucidation of LOS is pivotal to understanding the mechanisms of infection at the molecular level. The amphiphilic nature of LOS has been the main obstacle for structural analysis by matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry (MS). Our approach has resolved this important issue and has permitted us to obtain reflectron MALDI mass spectra of LOS to reveal the fine chemical structure with minimal structural variations. The high‐quality MALDI mass spectra show LOS species characteristic of molecular ions and defined fragments due to decay in the ion source. The in‐source decay yields B‐type ions, which correspond to core oligosaccharide(s), and Y‐type ions, which are related to lipid A unit(s). MALDI tandem time‐of‐flight (TOF/TOF) MS of lipid A allowed for the elucidation of its structure directly from purified intact LOS without the need for any chemical manipulations. These findings constitute a significant advancement in the analysis of such an important biomolecule by MALDI MS. Copyright © 2011 John Wiley & Sons, Ltd.