A systematic approach is described that can pinpoint the stereo-structures (sugar identity, anomeric configuration, and location) of individual sugar units within linear oligosaccharides. Using a highly modified mass spectrometer, dissociation of linear oligosaccharides in the gas phase was optimized along multiple-stage tandem dissociation pathways (MS(n), n = 4 or 5). The instrument was a hybrid triple quadrupole/linear ion trap mass spectrometer capable of high-efficiency bidirectional ion transfer between quadrupole arrays. Different types of collision-induced dissociation (CID), either on-resonance ion trap or beam-type CID could be utilized at any given stage of dissociation, enabling either glycosidic bond cleavages or cross-ring cleavages to be maximized when wanted. The approach first involves optimizing the isolation of disaccharide units as an ordered set of overlapping substructures via glycosidic bond cleavages during early stages of MS(n), with explicit intent to minimize cross-ring cleavages. Subsequently, cross-ring cleavages were optimized for individual disaccharides to yield key diagnostic product ions (m/z 221). Finally, fingerprint patterns that establish stereochemistry and anomeric configuration were obtained from the diagnostic ions via CID. Model linear oligosaccharides were derivatized at the reducing end, allowing overlapping ladders of disaccharides to be isolated from MS(n). High confidence stereo-structural determination was achieved by matching MS(n) CID of the diagnostic ions to synthetic standards via a spectral matching algorithm. Using this MS(n) (n = 4 or 5) approach, the stereo-structures, anomeric configurations, and locations of three individual sugar units within two pentasaccharides were successfully determined.