The cell envelope ofMycobacterium tuberculosis , the causative agent of tuberculosis in humans, is the source of carbohydrates of exceptional structure which play essential roles in the physiology of the bacterium and in its interactions with the host during infection. Much of what is known about their biosynthesis was derived from the phenotypic analysis of knockout or conditional knockout mutants of mycobacteria generated by random or specific insertional mutagenesis. Here, we describe the current techniques used to subfractionateM. tuberculosis cells and investigate major quantitative and qualitative changes in their cell envelope (lipo)polysaccharides.