Sterile filtration processes are widely used in the production of biotherapeutics for microorganism removal and product sterility. Sterile filtration processes can be applied to buffer preparation and cell culture media preparation in biotherapeutics processes, and to final sterilization or final filling in downstream processes. Owing to their broad range of applications in bioprocessing, various 0.2/0.22 μm sterile filters with different polymer materials (i.e., hydrophilic PVDF and PES) and nominal pore sizes are commercially available. The objective of this study was to evaluate two different commercial sterile filters in terms of filtration performance in various sterile filtration processes of biopharmaceutical production. The results demonstrate the importance of choosing the appropriate filter considering the process type and target removal/transport product to ensure efficient sterile filtration in the production of biotherapeutics.