Lentiviral vectors (LVs) are widely used in clinical trials of gene and cell therapy. Low LV stability incentivizes constant development and the improvement of gentle process steps. Steric exclusion chromatography (SXC) has gained interest in the field of virus purification but scaling up has not yet been addressed. In this study, the scaling up of lentiviral vector purification by SXC with membrane modules was approached. Visualization of the LVs captured on the membrane during SXC showed predominant usage of the upper membrane layer. Furthermore, testing of different housing geometries showed a strong influence on the uniform usage of the membrane. The main use of the first membrane layer places a completely new requirement on the scaling of the process and the membrane modules. When transferring the SXC process to smaller or larger membrane modules, it became apparent that scaling of the flow rate is a critical factor that must be related to the membrane area of the first layer. Performing SXC at different scales demonstrated that a certain critical minimum surface area-dependent flow rate is necessary to achieve reproducible LV recoveries. With the presented scaling approach, we were able to purify 980 mL LVs with a recovery of 68%.