This paper presents a new application of hollow fiber flow field-flow fractionation (HF5) as a preparative method to preconcentrate high mannose type N-linked glycoproteins from Streptococcus pyogenes by means of the mannose-specific binding affinity between concanavalian A (ConA) and N-linked glycosylated proteins. Prior to fractionation of N-linked glycoproteins from bacterial lysates, it was examined that ConA formed several types of multimers depending on the pH values (4, 6, and 8) of the carrier solution and it was confirmed that the molecular weight (MW) of ConA, spiked with alpha-1 acid glycoprotein (AGP) as a standard glycoprotein, increased due to binding with the mannose moiety of AGP. After adding ConA to bacterial lysates, mannose type N-linked glycoproteins were found to be enriched when the ConA fraction was isolated from whole bacterial lysates through HF5 run. For the identification of glycoproteins, the ConA fraction of HF5 was tryptically digested and followed by two-dimensional nanoflow strong cation exchange-reversed phase liquid chromatography-electrospray ionization-tandem mass spectrometry (2D SCX-RPLC-ESI-MS-MS) analysis to identify the N-linked glycoprotein species. From two-dimensional shotgun analyses, 45 proteins that exist on the Asn-Xaa-Ser/Thr sequence were identified as high mannose type N-linked glycoprotein. As a result, it was first demonstrated that HF5 is an alternative tool to enrich high mannose type N-linked glycoproteins using ConA-specific binding affinity.