Lipopolysaccharides (LPS) constitute the outermost layer of Gram-negative bacteria and consequently play an important role in bacterial infections. In order to address public health issues posed by Gram-negative bacteria, it is necessary to elucidate the structure of the molecular actors at the forefront of infections. LPS virulence and toxicity are partially modulated by lipid A, a hydrophobic saccharolipid that anchors LPS to the bacterial outer membrane. Understanding the lipid A structure is inherently intertwined with understanding its role as an endotoxin. Accordingly, several successful strategies incorporating tandem mass spectrometry have been applied toward the structural analysis of lipid A. Herein, a shotgun HCD strategy was applied toward the characterization of the lipid A profile of Pseudomonas aeruginosa PAO1. This analysis was enhanced by the development of an LC-MS/MS approach to eliminate isomeric signals in the MS/MS spectra that confounded characterization. Importantly, combining reverse phase chromatography with HCD and ultraviolet photodissociation analyses of the lipid A profile revealed the presence of previously unreported lipid A acyl chain positional isomers. Altogether, these strategies provide the most in-depth structural and molecular characterization of PAO1 lipid A to date.