Oligosaccharides have many isomers and MALDI-QIT-TOFMSn analysis is effective for determining their structures. However, it is difficult to elucidate in detail the structures of fucosylated and/or sialylated oligosaccharides that are known to be disease markers because fucose and sialic acid residues are easily released. We have introduced a technique of labeling oligosaccharides with a pyrene derivative prior to negative-ion MALDI-QIT-TOFMSn, and we have established a reliable method using this technique for the analysis of neutral oligosaccharides, such as fucosylated oligosaccharides containing blood group antigens H, Lea, and Lex. Intense and stable ionization in both positive and negative modes was achieved by derivatization with pyrene. As little as 10 fmol of pyrene-labeled oligosaccharides gave sufficient signals for analysis. Specific A-, D- or Y-type ions that depend on the structures of branching antennae could be detected by MSn and were useful for rapid and easy structural determination. These specific fragmentations resulting from collision-induced dissociation can be used to elucidate the structures of unknown oligosaccharides even if authentic oligosaccharides are not available as standards. By using this method, we identified and quantitated isomeric oligosaccharides with different fucosyl linkages from their mixtures. Moreover, sialylated oligosaccharide was converted to the corresponding neutral oligosaccharide by amidation, and the negative-ion spectrum was shown to be more informative than that of the original acidic oligosaccharide. Structural determination of both fucosylated and sialylated isomers, such as sialylfucosyllacto-N-hexaose I and monosialyl monofucosyllacto-N-neohexaose, was successful because fragment ions bearing fucose or amidated sialic acid were obtained on negative-MSn.