Hemolytic uremic syndrome caused by an invasive Streptococcus pneumoniae infection (SP-HUS) is a rare and severe disease that primarily affects children under two years of age. The pathophysiology of SP-HUS remains poorly understood, and treatment is largely supportive. Complement factor H (FH) is a key regulator of the alternative pathway of the complement system. It has been hypothesized that loss of sialic acids from FH’s N-glycans may impair its regulatory functions, thereby potentially leading to complement-mediated endothelial cell damage in SP-HUS. In this study, we investigated the N-glycosylation patterns of FH across three N-glycosylation sites for four SP-HUS patients and compared it to healthy controls using LC-MS/MS-based glycopeptide profiling.