Here we present Design of Experiment (DoE) strategy used to optimize and evaluate a number of different parameters for triple transfection, scalable and reproducible AAV upstream process for different serotypes, developed from small scale shake flask up to 25L in different single-use bioreactor systems. In addition, we present optimized and scalable downstream processing of AAV for high overall yields and efficient empty capsid product impurity removal. Furthermore, we present a surface plasmon resonance (SPR) assay for robust and accurate determination of virus titer.