Background Recombinant (rec-) coagulation factor VIII concentrates available for hemophilia A (HA) treatment differ in cell line production and structure, which could affect their pharmacodynamics and immunogenicity. Clinical trials showed that previously untreated patients with severe HA present higher rates of inhibitor development if treated with rec-FVIII products and that differences do exist as to inhibitor's formation among different rec-FVIII products. This finding could arise from several causes, such as absence of von Willebrand factor, different glycosylation profiles, or processes of molecular aggregation of the recombinant FVIII molecules. Objectives/Methods In this study, using size exclusion high-performance liquid chromatography (SE-HPLC), dynamic light scattering (DLS) spectroscopy, and functional biochemical assays, we investigated the purity grade, FX activating ability, and aggregation status of three recombinant marketed products (Advate [Baxalta], Refacto AF [Pfizer], and Kogenate [Bayer]). Results The overall analysis of the results obtained with SE-HPLC and DLS spectroscopy showed that the three recombinant FVIII concentrates contain low but significant amounts of molecular aggregates. This phenomenon was less evident for the Advate product. Molecular aggregation negatively affects the in vitro pharmacodynamics of the concentrates with higher aggregates' content. Conclusions This study shows that the three pharmaceutical formulations of recombinant FVIII contain variable amounts of molecular aggregates after their reconstitution at therapeutic concentrations. This phenomenon negatively affects the in vitro potency of the products with higher aggregates' content and might be invoked as a contributing cause of their increased risk to induce the formation of FVIII inhibitors.