Endocytosis is the most prevalent entry port for viruses into cells, but viruses must escape from the lumen of endosomes to ensure that viral genomes reach a site for replication and progeny formation. Endosomal escape also helps viruses bypass endo-lysosomal degradation and presentation to certain toll-like intrinsic immunity receptors. The mechanisms for cytosolic delivery of non-enveloped viruses or nucleocapsids from enveloped viruses are poorly understood, in part because no quantitative assays are readily available, which directly measure the penetration of viruses into the cytosol. Following uptake by clathrin-mediated endocytosis or macropinocytosis, the non-enveloped adenoviruses penetrate from endosomes to the cytosol, and they traffic with cellular motors on microtubules to the nucleus for replication. In this study, we present a novel single cell imaging assay, which quantitatively measures individual cytosolic viruses and distinguishes them from endosomal viruses or viruses at the plasma membrane. Using this assay, we show that the penetration of human adenoviruses from the species C and B occurs rapidly after virus uptake. Efficient penetration does not require acidic pH in endosomes. This assay is versatile, and can be adapted to other adenoviruses, and members of other non-enveloped and enveloped virus families.