A dual lectin-based size sorting and simultaneous enrichment strategy for selectively isolating N-linked glycopeptides was developed using asymmetrical flow field-flow fractionation (AF4). AF4 is an elution-based method for separating biological macromolecules that has been utilized for the separation of lectin-glycopeptide complexes formed by mixing serum peptides with lectin cocktails according to the difference in diffusion coefficients. It has also been used for simultaneous depletion of nonglycosylated peptides. The dual lectin-based enrichment method was applied to proteolytic peptides from lung cancer serum samples with two lectins (WGA, GlcNAc-specific, and SNA, Sia-specific), and the whole mixture was separated by AF4. The lectin-glycopeptide complex fractions collected during AF4 separation were endoglycosidically digested with PNGase F. The resulting deamidated glycopeptides were analyzed by nanoflow liquid chromatography-electrospray ionization-tandem mass spectrometry (nLC-ESI-MS-MS) to semiquantitatively profile the N-linked glycopeptides from the sera of lung cancer patients and healthy controls. The AF4 enrichment strategy coupled with nLC-ESI-MS-MS identified 16/24 (up/down-regulated by at least 10-fold compared to normal sera) N-linked glycopeptides from a WGA complex fraction of lung cancer sera and 18/3 from a SNA fraction.