A method for obtaining highly purified endotoxin (lipopolysaccharide [LPS]) in a few hours by repurification of commercial or laboratory preparations was devised. It avoids the use of phenol, which is not suitable for phenol-soluble lipopolysaccharides nor for some industrial purposes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization mass spectrometry analysis confirmed the integrity of the purified LPSs. The purified products did not activate Toll-like receptor 2 (TLR2), nuclear oligomerization domain 1 (NOD1), or NOD2 but did activate TLR4. Applied to different lipopolysaccharides, the method also improved their mass spectra, thus facilitating their structural analysis.