Lipopolysaccharide (LPS) is the signature glycolipid isolated from almost all Gram-negative bacteria. LPSs are well known for their ability to elicit the release of cytokines from eukaryotic cells including macrophages, neutrophils, and epithelial cells. LPS can be isolated free of contaminating nucleic acids and proteins by various techniques. In this review, we outline approaches for the isolation and preparation of LPSs for structural studies as well as preparation of very highly purified material for biological studies. Methods are also provided for the analysis of the purity and the structural composition of the LPSs. Finally, three methods for the isolation of lipid A are described.