Endotoxins (lipopolysaccharides) are the main components of Gram-negative bacterial outer membranes. A quick and simple way to isolate their lipid region (lipid A) directly from whole bacterial cells was devised. This method using hot ammonium-isobutyrate solvent was applied to small quantities of cells and proved to be indispensable when a rapid characterization of lipid A structure by mass spectrometry was required. Biological activities of endotoxins are directly related to the lipid A structures, which vary greatly with cell growth conditions. This method is suitable for rough- and smooth-type bacteria and very efficient for screening variations in lipid A structures. Data are acquired in a few hours and avoid the use of phenol in extraction.