Accurate quantification of viral vectors and vaccines is a crucial step required before any downstream use of virus preparations. The conventional immunocytochemistry-based method of adenovirus quantification has been widely used, but there are many areas for improvement toward accuracy and resource consumption savings to reduce viral miscalculation and wastage of vaccination materials. In this work, a one-step approach is implemented for optimized adenoviral quantification that uses a single antibody coupled with automated, high-throughput image acquisition and subsequent batch analysis. First, cells are infected with the adenovirus of interest and stained using the Hexon protein. Then, multichannel automated image acquisition via the Invitrogen EVOS M7000 Imaging System is performed. Last is an automated large batch analysis of acquired images via the EVOS Analysis Software, accomplished via precise training of the software allowing for virus infection quantification and additional parameters (counts, circularity, area and intensity of targets). It was found that the implemented approach yielded precise and accurate quantification of both oncolytic viral vaccines and gene therapy vectors in a time- and resource-effective manner when compared with conventional methodologies.