Oligosialic and polysialic acids (oligo/polySia) are characterized by high structural diversity, because of different types of sialic acids and glycosidic linkages. Although several methods have been described for the analysis of oligo/polySia, only high-performance liquid chromatography (HPLC) analysis in conjunction with 1,2-diamino-4,5-methylenedioxybenzene labeling, fluorometric C7/C9 detection, Western blotting, and matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF-MS) of lactonized oligo/polySia species, require submicrogram amounts of analyte. Since these methods do not provide detailed structural information, this study is focused on the characterization of oligo/polySia by tandem mass spectrometry (MS/MS). MALDI-TOF-MS/MS and electrospray ionization tandem mass spectrometry (ESI-MS/MS), employing up to three cycles of ion isolation and fragmentation in an ion trap, have been used for the characterization of nonderivatized glycans, oligoSia species modified at their reducing or nonreducing ends, as well as partially O-acetylated oligoSia derivatives. The obtained spectra were dominated by simultaneous cleavage of glycosidic linkages and the corresponding lactone ring, whereas classical cross-ring fragments were of minor abundance. However, the combined use of the two different types of fragmentation analysis allowed a sensitive and detailed characterization of both short-chained oligoSia and long polySia species. Furthermore, oxidation of the nonreducing end sugar moiety enabled sequence determination and localization of acetylated and nonacetylated sialic acid residues.