Epstein-Barr virus (EBV) infects various cell types in a wide spectrum of benign and malignant diseases. Laboratory tests for EBV have improved and are increasingly used in diagnosis, prognosis, prediction, and prevention of diseases ranging from infectious mononucleosis to selected subtypes of lymphoma, sarcoma, and carcinoma. Indeed, the presence of EBV is among the most effective tumor markers supporting clinical management of cancer patients. In biopsies, localization of EBER transcripts by in situ hybridization remains the gold standard for identifying latent infection. Other RNA- and protein-based assays detect lytic viral replication and can distinguish carcinoma-derived from lymphocyte-derived EBV in saliva or nasopharyngeal brushings. Analysis of blood using EBV viral load and serology reflects disease status and risk of progression. This review summarizes prior research in the context of basic virologic principles to provide a rational strategy for applying and interpreting EBV tests in various clinical settings. Such assays have been incorporated into standard clinical practice in selected settings such as diagnosis of primary infection and management of patients with immune dysfunction or nasopharyngeal carcinoma. As novel therapies are developed that target virus-infected cells or overcome the adverse effects of infection, laboratory testing becomes even more critical for determining when intervention is appropriate and the extent to which it has succeeded.