Replication defective adenoviruses have been used as vectors in a variety of settings including gene transfer, gene manipulation, and functionality studies. A quantitative real-time PCR-based assay is described for rapid determination of physical titers of recombinant adenovirus vectors. This method is based on amplification of a 77 bp fragment located near the left end of the adenovirus type 5 genome. Evaluation of this method demonstrated that it is simple, sensitive and reproducible, and has a dynamic range of quantitation over 5 logs. This assay is applicable to purified adenovirus as well as vectors prepared by simple cell lysis procedure, requiring only a small amount of starting material. The simplicity and short turn-around time of this assay should facilitate rapid titer determination for a large collection of adenoviral vectors.