Under certain circumstances, the determination of coagulation factor VIII (FVIII) is hampered by assay discrepancies between clotting and chromogenic approaches. These are observed in certain patients’ plasma as well as in certain concentrates. We intended to develop a novel assay for the quantification of coagulation FVIII which reflects the physiological situation better than the established assays. It is based on plasma without chelation of divalent cations and simultaneously minimizes the generation of activated factors which could function as uncontrolled triggers of coagulation. FVIII deficient plasma is prepared with the aid of biotinylated antibodies against FVIII from normal plasma in presence of inhibitors of contact activation. To start the assay only tiny amounts of activated FIX serve as trigger. The FVIII determination is performed in a kinetic experiment and is based on the cleavage of a fluorogenic substrate for activated FX. FVIII concentrations between 0.01 and 1 IU mL−1 are easily determined. Plasma-derived and recombinant FVIII concentrates were compared. All plasma-derived concentrates were found to contain FVIII activities within the specification of the manufacturer. Recombinant concentrates yielded only 35–50% of the claimed potency. The novel in vivo-like assay avoids the undue advantage or disadvantage of certain product characteristics by eliminating unphysiological assay conditions. Its usefulness could turn out in future experiments with plasma from haemophilia A patients.