We describe a simple and rapid assay for determining the titer of functional and infectious adenoviruses that yields reliable results within 36 h. The method consists of an initial incubation of serial diluted adenoviruses on HEK293 cells and a subsequent detection of adenovirus genomic DNA with quantitative polymerase chain reaction. With an adenovirus of known titer as a reference, the exact titers of four unknown adenoviruses could be easily and accurately determined in one 96-well plate. This method can be potentially modified for quality control of other viruses.