Structural isomers of sialylated N‐glycans contribute to the diversity of the N‐glycome and to a range of biological functions. Sialyl linkage isomers can be readily distinguished by mass spectrometry with mass differences between α2,3‐ and α2,6‐linkages generated by a two‐step sialic acid linkage‐specific alkylamidation. To improve the identification of N‐glycans from complex mixtures, we added a delactonization step after the first alkylamidation step, which regenerates negatively charged carboxylic acids on α2,3‐sialic acids. N‐glycan isomers with α2,3‐sialic acids are then fractionated by ion‐exchange chromatography prior to the second alkylamidation step. With this modified alkylamidation method, sialylated N‐glycans were enriched and stabilized for structural characterization by capillary electrophoresis–mass spectrometry and tandem mass spectrometry. We identified 52 sialylated N‐glycan structures, including 107 linkage isomers, in human serum and confirmed the presence of positional isomers of specific sialyl linkage isomers. Due to the reduced sample complexity after ion‐exchange fractionation and CE separation, substructural features of N‐glycans were rapidly evaluated and included core‐ and antenna‐fucosylation and poly‐lactosamine.