CDC researchers have developed a real-time PCR assay for the detection and viral-load quantitative estimations of human bocavirus (HBoV) from clinical specimens. At present, there have been few reports on the epidemiology, geographic distribution or clinical features of HBoV infection. Additionally, symptoms affiliated with bocavirus infections overlap with numerous other respiratory illnesses. This CDC assay provides sensitive, specific, and quantitative detection of HBoV in patients with respiratory illness by a method of real-time PCR targeting the HBoV NS1 and NP-1 genes. Use of this assay in conjunction with additional diagnostic methods and treatments should facilitate improved diagnosis and, subsequently, directed treatment and patient outcome.