Scaling bioprocesses remains a major challenge. Since it is physically impossible to increase all process parameters equally, a suitable scale-up strategy must be selected for a successful bioprocess. One of the most widely used criteria when scaling up bioprocesses is the specific power input. However, this represents only an average value. This study aims to determine the Kolmogorov length scale distribution by means of computational fluid dynamics (CFD) and to use it as an alternative scale-up criterion for geometrically non-similar bioreactors for the first time. In order to obtain a comparable Kolmogorov length scale distribution, an automated geometry and process parameter optimization was carried out using the open-source tools OpenFOAM and DAKOTA. The Kolmogorov−Smirnov test statistic was used for optimization. A HEK293-F cell expansion (batch mode) from benchtop (Infors Minifors 2 with 4 L working volume) to pilot scale (D-DCU from Sartorius with 30 L working volume) was carried out. As a reference cultivation, the classical scale-up approach with constant specific power input (233 W m−3) was used, where a maximum viable cell density (VCDmax) of 5.02·106 cells mL−1 was achieved (VCDmax at laboratory scale 5.77·106 cells mL−1). Through the automated optimization of the stirrer geometry (three parameters), position and speed, comparable cultivation results were achieved as in the small scale with a maximum VCD of 5.60·106 cells mL−1. In addition, even on the pilot scale, cell aggregate size distribution was seen to strictly follow a geometric distribution and can be predicted with the help of CFD with the previously published correlation.