Abstract: Applications of adeno-associated virus (AAV) in gene therapy has increased significantly in the last two decades due to its remarkable safety profiles and efficiency of gene delivery into various tissues. Demand on recombinant AAV vectors has also increased to fulfill pre-clinical and clinical requirements. Helpervirus-free mediated transient transfection has been widely adopted. Low titer and process scalability remain two key issues in AAV manufacturing. The productivity of AAV vectors is often affected by the AAV serotype, cell culture type, the size of Gene of Interest (GOI), cell density, transfection method, plasmid DNA concentration and other factors. Based on the risk assessment of these factors that may impact AAV production with helper-virus-free mediated transient transfection, we have evaluated 6 process parameters using JMP’s Definitive Screening Design. In this study, critical process parameters (CPP) were identified as plasmid DNA to cell density ratio, cell density per surface area and incubation time post transfection. Their operating ranges were established for optimum and robust AAV production, which can be applied while scaling up to manufacturing. AAV expression level was improved to 2E11 vg/mL.