Recombinant adeno-associated viral vectors (rAAV) are the vehicle of choice for therapeutic gene delivery in the gene therapy field. The transient transfection method of production remains one of the most attractive processes for the manufacture of AAV due to its fast-turnaround and versatility towards the production of a wide-range of rAAV constructs. To meet the increasing drug substance demand, further productivity improvement has been one of the primary focuses of process development. Polyethylenimine (PEI) is the most popular transfection reagent for manufacturing of rAAVs at a large scale due to being highly efficient, stable for long term storage, and economically feasible. To address the well-known dilemma between transfection efficiency and PEI induced cytotoxicity, research efforts have been focused on areas including identifying the optimal PEI structure and mol. weight, chem. engineering novel PEI derivative, or searching for alternative cationic polymers. However, com. availability and lot-to-lot consistency limit the choice of GMP compliant PEI sourced for manufacturing scale production To maximize the efficiency vs. cytotoxicity ratio of a particular PEI, critical process parameters including cell d., polyplex addition, DNA/PEI ratio, medium and additives, as well as dissolved oxygen were identified and optimized comprehensively. This study has confirmed ROS is one main root cause of the adverse effects of PEI and can result in a dramatic AAV productivity decrease. We have also demonstrated the effectiveness of achieving more than 10 folds of AAV productivity improvement through independent or orthogonal process changes focusing on reducing ROS