Significant increases in cell density and product titer have led to renewed interest in the application of depth filtration for initial clarification of cell culture fluid in antibody production. The performance of these depth filters will depend on the local pressure and velocity distribution within the filter capsule, but these are very difficult to probe experimentally, leading to challenges in both process design and scale‐up. We have used a combination of carefully designed experimental studies and computational fluid dynamics (CFD) to examine these issues in both lab‐scale (SupracapTM 50) and pilot‐scale (StaxTM) depth filter modules, both employing dual‐layer lenticular PDH4 media containing diatomaceous earth. The SupracapTM 50 showed a more rapid increase in transmembrane pressure and a more rapid DNA breakthrough during filtration of a Chinese Hamster Ovary cell culture fluid. These results were explained using CFD calculations which showed very different flow distributions within the modules. CFD predictions were further validated using measurements of the residence time distribution and dye binding in both the lab‐scale and pilot‐plant modules. These results provide important insights into the factors controlling the performance and scale‐up of these commercially important depth filters as well as a framework that can be broadly applied to develop more effective depth filters and depth filtration processes. The authors evaluated the performance characteristics for both lab‐and pilot‐scale depth filtration modules for Chinese Hamster Ovary (CHO) cell clarification. The pilot‐scale module showed greater capacity and DNA removal due to more uniform flow distribution, which was verified by computational fluid dynamics, residence time distribution, and dye‐binding studies.