Summary Congenital Hemophilia A (HA) is an X-linked bleeding disorder due to a lack or dysfunction of coagulation FVIII (FVIII). Nearly 1 in 5,000 live male births are diagnosed with HA with 40% of whom involving of sporadic cases caused by de novo mutations. Plasma-derived FVIII (pdFVIII) and recombinant FVIII (rFVIII) are two major intravenous replacement therapy medicines for HA treatment. FVIII neutralizing antibodies, or inhibitors, occur in 25-30% of severe HA patients receiving protein replacement. These FVIII inhibitors essentially abolish the efficacy of the coagulant treatment, resulting in a considerable increase in the morbidity and healthcare cost. Although both rFVIII and pdFVIII share similar to identical primary amino acids, the risk of FVIII antibodies in previously untreated patients (PUPs) was reported to be higher following rFVIII treatment. As a glycoprotein, pdFVIII and rFVIII are different in glycosylation, a post-translation modification (PTM) that is capable of alter the immunogenicity of protein Therefore, in this project, we will develop a set of novel glycoanalysis toolset for FVIII inhibitors. In order to investigate the effect of the host glyco environment on FVIII, we propose to use FVIII closely associated VWF and FV as surrogate marker to show to potential effect on FVIII glycosylation in HA patients who developed FVIII inhibitors. Also, based on our developed MS-based approach, glycosylation pattern of total immunoglobulins, specific FVIII inhibitors, FV, VWF, and overall glycome changes of the host cell environment will be thoroughly characterized in a large number of HA patients’ samples, close attention to those undergone immune tolerance induction (ITI). The overall results obtained from this project will be significant for understanding the fundamental mechanism of FVIII immunogenicity.