Virus filtration is a robust component of the downstream purification process used for production of important biotherapeutics. Several recent studies have shown that fluorescently-labeled bacteriophage can be used to study virus capture during virus filtration, but these bacteriophage are available with only a relatively limited size range and the preparation, purification, and labeling of these model viruses is challenging. The objective of this study was to demonstrate the potential of using confocal laser scanning microscopy with fluorescently-labeled nanoparticles of different size ( approximately 20, 40, and 100 nm) to study the internal pore structure and virus capture behavior within different virus filters. Filtration experiments were performed using relatively homogeneous Ultipor((R)) DV20 membranes and highly asymmetric Viresolve((R)) Pro membranes. The capture patterns seen with 20 nm nanoparticles were similar to images obtained using labeled bacteriophage, validating the use of these fluorescent nanoparticles as surrogates for small parvovirus. Confocal images obtained with different size nanoparticles provided insights into the internal gradient in pore size within the different virus filters. These results provide new insights into the morphology of commercial virus filters while demonstrating the effectiveness of using fluorescent nanoparticles to study virus capture and retention.