Two-dimensional high-performance liquid chromatography using an electrospray ionization time-of-flight mass spectrometry (2D-HPLC-ESI-TOF-MS) system was established for the on-line determination of asparagine-linked oligosaccharides in glycopeptides. The analysis of the oligosaccharides started with the enzymic transglycosylation reaction utilizing Endo-beta-N-acetylglucosaminidase (Endo-M). The oligosaccharides were transferred to a fluorescent acceptor (NDA-Asn-GlcNAc) with Endo-M to produce the fluorescent oligosaccharides. The resulting fluorescent oligosaccharides were specifically isolated from the non-fluorescent oligosaccharides with fluorescence detection after separation by the 1st dimension Amide-80 column. The fraction of fluorescent oligosaccharides was effectively trapped in the anion exchange column. The trapped oligosaccharides were then separated by the 2nd dimension ODS column and sensitively determined by ESI-TOF-MS. Disialo-Asn (a model oligosaccharide) and several oligosaccharides liberated from ovalbumin could be efficiently separated by the 2D-HPLC and identified from the ESI-TOF-MS. Based on these results, the proposed 2D-HPLC-ESI-TOF-MS system may be useful for on-line oligosaccharide analyses. Although the analytical run time is still long, a high-throughput determination will be performed by optimization of the 2D-HPLC conditions.