The assessment of recombinant FVIII (rFVIII) activity (FVIII:C) in plasma of patients is dependent on the assay. Notably, a calibration with a product-specific laboratory standard is recommended when measuring Refacto-AFR activity in plasma with a one-stage assay. The objective of this study was to facilitate the measurement of rFVIII, taking into account the recent demonstration that a calibration curve does not have to be included in each run. FVIII:C was measured in patients' samples after infusion of different types of rFVIII with a one-stage and a chromogenic assay calibrated either with pooled normal plasma or a product-specific laboratory standard. Results obtained with the one-stage coagulation assay were compared with these provided by a chromogenic assay. We confirmed that a calibration curve can be used for a prolonged period of time without loss of precision and accuracy. In such conditions, a stable relation between the calibration curves generated with a product-specific laboratory standard and plasma can be established. In patients' plasma, Refacto-AF levels measured with a one-stage FVIII assay calibrated with plasma or a product-specific laboratory standard diverged from −58% to −17% and from −25% to +18%, respectively, from the activity determined with a chromogenic substrate assay. By comparison, FVIII:C levels of full-length rFVIII measured with the one-stage assay calibrated with plasma were 6–49% lower than with the chromogenic assay. In a monocentric setting, the long-term stability of the calibration curves allows the implementation of a practical and cost-effective approach to determine rFVIII:C levels.