Accurate measurements of the physical and infectious titers of adenoviral vectors are crucial for evaluating preclinical studies and for the safety and efficacy of clinical studies. Unfortunately, there are no standardized methods of measurement, consequently variable and unreliable values are the result. Furthermore, infectious titers of helper-dependent adenoviral vectors (HDAd) are difficult to measure because traditional cytopathic effect assays cannot be employed, thus hindering their potential clinical application. In response to this problem, a fully characterized Adenovirus Reference Material (ARM) has been developed to be used as a reference standard for clinical grade adenoviral vectors. However, no specific protocols for this purpose have been provided. To fulfill this important need, we have developed a simple assay involving co-infection of 293 cells with the adenoviral vector and the ARM to permit direct comparisons of their physical and infectious titers. We demonstrate, using a HDAd, that this co-infection assay is reliable, sensitive, and reproducible. Importantly, this assay is inherently unaffected by variables that plague other methods of determining vector titers. This assay is applicable to all human serotype 5 adenoviral vectors and will permit reliable comparisons within and between studies as well as meet an important prerequisite for clinical studies.