Imaging host–pathogen interactions in real time can provide significant insight into dynamic processes and provide information about time and space of their occurences. Here, we present detailed experimental instructions on how to image the membrane penetration process of the non-enveloped adenovirus in real time. The system is based on a cell line stably expressing the lectin galectin-3 fused to a fluorophore. Membrane-lytic events during adenovirus cell entry can be monitored by the recruitment of galectin-3 to galactose-containing membrane glycoproteins on the exo-surface of ruptured membranes. The simultaneous use of fluorescently labeled adenoviral capsids allows to image the events in unmatched temporal resolution.