Adeno-associated virus (AAV)-based gene therapies can restore endogenous factor VIII (FVIII) expression in hemophilia A (HA). AAV vectors typically utilize a B-domain deleted FVIII transgene, such as human FVIII-SQ in valoctocogene roxaparvovec (AAV5-FVIII-SQ). Surprisingly, the activity of transgene-produced FVIII-SQ was between 1.3 and 2.0 times higher in one-stage clot (OS) than chromogenic-substrate (CS) assays, while recombinant FVIII-SQ products have lower OS than CS activity. Transgene-produced and recombinant FVIII-SQ showed comparable specific activity (IU/mg) in the CS assay, demonstrating that the diverging activities arise in the OS assay. Higher OS activity for transgene-produced FVIII-SQ was observed across various assays kits and clinical laboratories, suggesting intrinsic molecular features as a potential root cause. Further experiments in two participants showed that transgene-produced FVIII-SQ accelerated early factor Xa and thrombin formation, which may explain the higher OS activity based on a kinetic bias between OS and CS assay readout times. Despite the faster onset of coagulation, global thrombin levels were unaffected. A correlation with joint bleeds suggested that both OS and CS assay remained clinically meaningful to distinguish hemophilic from non-hemophilic FVIII activity levels. During clinical development, the CS activity was chosen as a surrogate endpoint to conservatively assess hemostatic efficacy and enable comparison with recombinant FVIII-SQ products. Relevant trials are registered on Clinicaltrials.gov (NCT02576795 and NCT03370913).