Cell therapies based on inducible pluripotent stem cells offer promising new treatments for a variety of different illnesses. However, the sensitivity of stem cells to hydrodynamic stress makes developing reliable stem cell production processes challenging. Understanding hydrodynamic stress conditions experienced by stem cells during early-stage process development is important to guide scale-up and design scale-down experiments. We characterize the hydrodynamic stresses in a 125 mL shake flask using Lattice-Boltzmann implicit large eddy simulations (LB-ILES). First, we validated the LB-ILES shake flask simulations using volumetric power input measurements and experimental liquid distribution data showing good overall agreement, while also numerical challenges of the LB-ILES method regarding grid and time step dependencies are discussed. The mean shear stress in the shake flask increases from 0.01 to 0.24 Pa when increasing the shaking frequency from 55 to 250 rpm, and the mean Kolmogorov length scale decreases from 185 to 51 μm. Furthermore, time-averaged distributions of the shear stress and Kolmogorov length scales were evaluated and compared to reported stress thresholds for stem cells. Based on the shear stress and Kolmogorov length scale distributions, our developed shake flask CFD model can help to design small-scale experiments to characterize stem cell cultures in terms of their hydrodynamic stress tolerance, and ultimately guide scale-up stem cell cultures to larger cultivation systems.