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Abstract: During the cell culture in stirred aerated vessels, both stirring and sparging is required for proper homogenization of a mixture and oxygen delivery. Quality by design (QbD) in biopharmaceutical industry aims at identification of the impact of mass transfer, dissolved oxygen, pH and hydrodynamic stress on the cell behavior, productivity and product quality. Most of these properties are influenced by impeller motion and sparger aeration. In this research, the shear sensitive poly(methyl methacrylate) (PMMA) aggregates are used as probes capable of determining the maximum hydrodynamic stress (τmax) present in the ambr250® bioreactor. The results from our micro-aggregate probes are connected with the cultivation data using two Chinese hamster ovary K1 glutamine synthetase mediated cell lines (CHO-K1 GS) to determine the stress threshold, below which no significant impact of operating conditions on cell behavior is observed. Such calibrated system can be in the future used as a scale-down model of large-scale bioreactors to test cell robustness during process development.