High-mannose and hybrid-type N-glycans are present in human serum glycoproteins in low abundance but have recently been described to play an important role in immune responses. It is therefore important to find a strategy to selectively analyze their structures in the context of health and disease in order to understand their impact on disease mechanisms. We report here the characterization of high-mannose and hybrid-type N-glycans in total human serum. To this end, N-glycans were released using Endo-beta-N-acetylglucosaminidase H (Endo H) and analyzed by CE-LIF and MALDI-TOF-MS. We found that the high-mannose structures Man59GlcNAc1 represented the majority of the pool. The monoglucosylated structure Glc1Man9GlcNAc1 as well as four hybrid structures could be identified. Then, we compared the Endo H-released serum glycome of patients suffering from rheumatoid arthritis with healthy controls as mannose-binding lectin deficiency (MBL) and modulation of a-mannosidase activity were previously associated with this disease. Interestingly, we observed that both high-mannose and hybrid structures were fairly constant, suggesting that circulating MBL and a-mannosidase may not affect significantly the levels of serum glycoproteins carrying these glycans.