Setting up robust and reproducible bioprocesses is a top priority for all biotechnology companies focused on developing life changing AAV-based gene therapies. To do so, innovators crave for breakthrough technologies to maximize productivity while controlling the cost of goods (CoGs) of their AAV vector manufacturing workflow, key to accelerate the speed to commercialization of their clinical programs pipeline. Helper-free triple plasmid transfection of HEK293 cells – adherent or suspension – is the most used upstream method for AAV manufacture. Observing consistent plasmid DNA delivery among multiple production runs is critical but requires some effort as finding optimal transfection conditions for a given bioprocess is not plug and play. Still, significant advances have been recently made towards standardization of the triple transfection process. Here, we discuss how optimizing the transfection step during upstream process development leverages tremendous benefits on overall costs, robustness and scalability of an AAV production platform.