Lipids are essential components of all bacterial membranes. The most common membrane-associated lipids in Gram-negative bacteria are phospholipids and lipid A, the hydrophobic anchor of lipopolysaccharide. Diversity in these lipids arises through structural modifications that include changes in the length and location of fatty acids, and the addition of phosphate and carbohydrate moieties. Analysis of individual structural modifications normally requires large quantities of starting material and multiple methods for the isolation, hydrolysis, and analysis. In this study, we developed a novel one-step protocol for the combined isolation of phospholipids and lipid A from Francisella subspecies followed by analysis using matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry. The total time for lipid isolation and analysis was approximately 15 min and with a lower limit of detection of approximately 100 ng of purified lipid. This protocol identified the major lipid structures using both wild-type Ft subspecies strains and lipid A biosynthesis mutants. We also determined the relative levels of individual lipid A and phospholipids after growth under conditions that mimic the mammalian infection process. This analysis showed that the bacterial membranes remodeled rapidly to adapt to changes in environmental growth conditions and may be important for Francisella pathogenesis. Copyright © 2011 John Wiley & Sons, Ltd.