AbstractHaptoglobin (Hp) is a positive acute phase protein, synthesized in the liver, with four N‐glycosylation sites carrying mainly complex type N‐glycans. Its glycosylation is altered in different types of diseases but still has not been extensively studied mainly due to analytical challenges, especially the lack of a fast, efficient, and robust high‐throughput Hp isolation procedure. Here, we describe the development of a high‐throughput method for Hp enrichment from human plasma, based on monolithic chromatographic support in immunoaffinity mode and downstream Hp N‐glycome analysis by hydrophilic interaction ultrahigh‐performance liquid chromatography with fluorescent detection (HILIC–UHPLC–FLR). Chromatographic monolithic supports in a 96‐well format enable fast, efficient, and robust Hp enrichment directly from diluted plasma samples. The N‐glycome analysis demonstrated that a degree of Hp deglycosylation differs depending on the conditions used for N‐glycan release and on the specific glycosylation site, with Asn 241 being the most resistant to deglycosylation under tested conditions. HILIC–UHPLC–FLR analysis enables robust quantification of 28 individual chromatographic peaks, in which N‐glycan compositions were determined by UHPLC coupled to electrospray ionization quadrupole time of flight mass spectrometry. The developed analytical approach enables fast evaluation of total Hp N‐glycosylation and is applicable in large‐scale studies.