Cell culture scale‐up is a challenging task due to the simultaneous change of multiple hydrodynamic process characteristics and their different dependencies on the bioreactor size as well as variation in the requirements of individual cell lines. Conventionally, the volumetric power input is the most common parameter to select the impeller speed for scale‐up, however, it is well reported that this approach fails when there are huge differences in bioreactor scales. In this study, different scale‐up criteria are evaluated. At first, different hydrodynamic characteristics are assessed using computational fluid dynamics data for four single‐use bioreactors, the Mobius® CellReady 3 L, the Xcellerex™ XDR‐10, the Xcellerex™ XDR‐200, and the Xcellerex™ XDR‐2000. On the basis of this numerical data, several potential scale‐up criteria such as volumetric power input, impeller tip speed, mixing time, maximum hydrodynamic stress, and average strain rate in the impeller zone are evaluated. Out of all these criteria, the latter is found to be most appropriate, and the successful scale‐up from 3 to 10 L bioreactor and to 200 L bioreactor is confirmed with cell culture experiments using Chinese Hamster Ovary cell cultivation.