ipid A, the hydrophobic anchor of lipopolysaccharide (LPS) is a potent sepsis-inducing molecule that stimulates human immune responses. The structural diversity of lipid A is correlated both with pathogenic capability and adaptation to the unique bacterial growth environments. Thus, understanding the structure of lipid A is important in fundamental microbiology and host-pathogen interactions. Previously, our group developed Fast Lipid Analysis Technique (FLAT) which employs an on-surface extraction combined with conventional Matrix-Assisted Laser Desorption/Ionization Mass Spectrometer (MALDI-MS), allowing for rapid structural characterization of lipid A directly from raw biomass of bacterial cells. Here, we employ tandem MS in both polarities, negative-ion (FLATn) and positive-ion (FLATn+) modes, to elucidate the structure of lipid A of various Gram-negative bacteria form single colonies.