A critical step in working with adenovirus (Ad) and its vectors is the accurate, reproducible, sensitive, and rapid measurement of the amount of virus present in a stock. Titration methods fall into one of two categories: determination of either the infectious or the particle (infectious plus noninfectious) titer. Determining the infectious titer of a virus stock by plaque assay has important limitations, including cell line-, researcher-, and laboratory-dependent variation in titer, and the length of time required to perform the assay (2–4 wk). A major drawback of particle titration methods is the lack of consistent correlation between the resultant titer and the infectious titer. To overcome these problems, a rapid, sensitive, and reproducible real-time polymerase chain reaction (PCR) assay was developed that detects encapsidated full-length genomes. Importantly, there is a linear correlation between the titer determined by the real-time PCR assay and the infectious titer determined by a plaque assay. This chapter provides step-by-step guidance for preparing viral DNA, conducting the real-time PCR assay, and using the resultant data to calculate a viral titer.