Structural characterization of mono-, di-, and trisulfated trisaccharides by fast atom bombardment (FAB) and electrospray ionization (ESI) mass spectrometry is described. Both FAB and ESI collision-induced dissociation tandem mass spectrometry (CID-MS/MS) of sulfated trisaccharides allowed identification of the sulfated sugar and provided information on the saccharide chain sequence. The ESI CID-MS/MS of multiple-charged ions provided even more structural information, and some of the useful daughter ions appeared in the higher mass region than the precursor owing to a lower charge-state. These methodologies can be applied to the structural analyses of glycoconjugates with much larger molecular masses and higher polarity, such as the poly-sulfated analogs.