In this chapter, we describe in detail the steps involved in isolation and characterization of lipoglycans from Mycobacterium tuberculosis and Mycobacterium smegmatis. In addition, procedures involved in structural analysis such as immunoblotting with mAb CS-35 or CS-40, gas chromatography, gas chromatography/mass spectrometry, nuclear magnetic resonance spectroscopy, and endoarabinanase digestion followed by high-pH anion exchange chromatography and two-dimensional gel electrophoresis are presented.