Accurate and precise potency determination by manufacturers of different types of factor VIII product (plasma-derived and recombinant FVIII [rFVIII]) is vital to clinicians and patients using FVIII concentrates. A separate, but related, requirement is ascertaining the FVIII activity levels in clinical samples for diagnosing and treating hemophilia A. The one-stage clotting assay (OSA) and the chromogenic substrate assay (CSA) are the main assays used for these measurements, with both assays being used for potency assignments, and the OSA also being widely used for clinical monitoring. Although the assays can produce concordant results, discrepancies often occur, e.g. when measuring FVIII levels in patients with mild or moderate hemophilia A, or when assaying high-purity FVIII products. Modifications to rFVIII proteins, such as B-domain deletion (BDD), and technologies for improving the pharmacokinetic profile of rFVIII may exacerbate assay discrepancies. The CSA appears to be essentially unaffected by these modifications. However, the OSA underestimates the FVIII activity levels and therapeutic potential of some further modified BDD rFVIII products, especially those conjugated to poly(ethylene glycol); the extent of the effects is dependent on the specific OSA reagents used. Although the OSA remains the preferred choice for clinical monitoring in Europe and the USA, an awareness of the limitations of that assay has prompted more laboratories to adopt the CSA.