Human cytomegalovirus (CMV), also known as human herpes virus-5 (HHV-5), is a common human pathogen acquired early in life in the majority of immunocompetent individuals. Primary infection establishes a state of latency and the virus can be reactivated during immunosuppression. CMV is a significant cause of morbidity and mortality in newborns and patients with impaired immune system. Prenatal infection can result in intrauterine growth retardation, hepatitis, myocarditis, pneumonitis, and neurologic abnormalities. Individuals with congenital or acquired immunosuppression can develop a primary CMV infection, infection with another CMV strain or experience reactivation of the latent virus. The hematopoietic stem cell and solid organ transplant recipients are at high risk of developing CMV infection, especially early in a post-transplant period. The definition of CMV disease includes the evidence of end-organ involvement in the presence of CMV detected by a validated laboratory assay. The selection of a laboratory method is highly dependent on the type of sample to be tested and the clinical presentation. In the clinical practice, the quantitative PCR-based assays are most helpful, since they can measure the level of CMV DNA in whole blood, plasma, cerebrospinal fluid, amniotic fluid, tissue, and urine, and follow the kinetics of infection. In this chapter we describe the PCR assay designed to quantify CMV DNA in human plasma by amplifying a 105 base-pair (bp) fragment of the CMV immediate-early DNA polymerase gene.