Perfusion cell culture technology has gained a lot of interest in recent years in the biopharmaceutical industry. One common application is N-1 perfusion which is used to intensify fed batch production processes and increase facility output. Upon running our perfusion process for the first time at manufacturing scale, unexpected cell damage was observed. Reducing the recirculation pump speed resulted in improvements in cell viability which implied the impact of pump shear stress on cell viability. In this study, we used polymethyl methacrylate (PMMA) nanoparticles to determine the shear stress inside two different sized rotary lobe pumps used in N-1 perfusion. The results were used to validate a computational fluid dynamics (CFD) model to predict the maximum shear under different operating conditions of the pump. The CFD model identified the radial and mesh clearance zones as regions that experience the maximum shear stress inside the pump. The model was then used to evaluate the impact of different geometry modifications in the pump lobes, and it predicted a 17% reduction in the maximum shear stress by increasing the mesh and radial clearances by 0.08 mm and 0.13 mm, respectively. The study indicates that CFD can be a useful tool to predict shear stress inside rotary pumps. The results can be used to optimize the pump operating conditions or even customize the pump geometry to save time and cost of process scaling to manufacturing without compromising the preset operating conditions or critical scale-up parameters.