Modification of structures of lipooligosaccharides (LOS) represents one prevalent mechanism by which Gram-negative bacteria can become resistant to key antibiotics. Owing to the significant complexity of LOS, the structural characterization of these amphipathic lipids has largely focused on elucidation of the lipid A substructures. Analysis of intact LOS enables detection of core oligosaccharide modifications and gives insight into the heterogeneity that results from combinations of lipid A and oligosaccharide substructures. Top-down analysis of intact LOS also provides the opportunity to determine unknown oligosaccharide structures, which is particularly advantageous in the context of glycoconjugate vaccine development. Advances in mass spectrometry technologies, including the development of MSn capabilities and alternative ion activation techniques, have made top-down analysis an indispensable tool for structural characterization of complex biomolecules. Here we combine online chromatographic separations with MS3 utilizing ultraviolet photodissociation (UVPD) and higher-energy collisional dissociation (HCD). HCD generally provides information about the presence of labile modifications via neutral loss fragments in addition to the saccharide linkage arrangement, whereas UVPD gives more detailed insight about saccharide branching and the positions of nonstoichiometric modifications. This integrated approach was used to characterize LOS from Acinetobacter baumannii 1205 and 5075. Notably, MS3 analysis of A. baumannii 1205, an antibiotic-resistant strain, confirmed phosphoethanolamine and hexosamine modification of the lipid A substructure and further enabled derivation of a core oligosaccharide structure.