Chemically defined and animal derived free components media are required for bioprocess development and operation using mammalian cell as platform for the production of biopharmaceuticals. The supplementation of commercial chemically-defined media with specific compounds is studied for the optimization of CHO and HEK 293 cell culture. Design of Experiments approach is used to first screen the most efficient compounds and further determine their optimal conditions. This methodology allows to test simultaneously various compounds and also to determine potential interaction among them, additionally to their individual effects. Using this methodology, the culture of CHO S cell growth could be improved by using a supplementation of selenium (1 µg/L), r-transferrin (20.2 mg/L) and r-insulin (26.1 mg/L). Further, HEK 293 cells could also be improved both in cell growth and also transfection efficiency using FreeStyle medium supplemented with CHO S cells, the optimal conditions were found for FreeStyle medium supplemented with r-insulin (19.8 mg/L), r-transferrin (1.6 mg/L) and lipid mix (0.9X).