The assembly of the enzyme activated Factor IX (FIXa) with its cofactor, activated Factor VIII (FVIIIa) is a crucial event in the coagulation cascade. The absence or dysfunction of either enzyme or cofactor severely compromises hemostasis, and causes hemophilia. FIXa is a notoriously inefficient enzyme, and needs FVIIIa to drive its hemostatic potential, by a mechanism that has remained largely elusive to date. In this study we employed Hydrogen-Deuterium eXchange-Mass Spectrometry (HDX-MS) to investigate how FIXa responds to assembly with FVIIIa in presence of phospholipids. This revealed a complex pattern of changes that partially overlaps with those that occur upon occupation of the substrate-binding site by an active site-directed inhibitor. Among the changes driven by both cofactor and substrate, HDX-MS highlighted several surface loops that have been implicated in allosteric networks in related coagulation enzymes. Inspection of FVIIIa-specific changes indicated that three helices are involved in FIXa-FVIIIa assembly. These are part of a basic interface that is also known as Exosite II. Mutagenesis of basic residues herein, followed by functional studies, indeed identified this interface as an extended FVIIIa-interactive patch. HDX-MS was also applied to recombinant FIXa variants that are associated with severe hemophilia B. This revealed that single amino acid substitutions can silence the extended network of FVIIIa-driven allosteric changes. We conclude that HDX-MS has the potential to visualize the functional impact of disease-associated mutations on enzyme-cofactor complexes in the hemostatic system.