The human adenovirus (hAdV) group is represented by 52 serotypes that have been reported to cause a broad range of clinical manifestations including respiratory tract infections, acute conjunctivitis, cystitis, gastroenteritis, and systemic infections. Conventional methods for detection of hAdVs include electron microscopy, antigen detection, and virus isolation in cell culture. Implementation of real-time PCR assays has increased the sensitivity and speed of detection, and allowed for rapid quantification and serotyping. This chapter describes the design and validation of a multiplex real-time PCR assay for the detection of a broad range of hAdV serotypes in respiratory samples, blood, or urine. This assay targets the conserved region of the hAdV hexon gene and utilizes hydrolysis probes for the detection of amplified products. The assay can be adapted to provide quantitative results to evaluate the change in viral load, and products can be sequenced for serotype designation. PCR-based methods for hAdV detection are sensitive, specific, allow for rapid diagnosis, and facilitate epidemiological studies.