BACKGROUND Epstein-Barr virus (EBV) is a human herpesvirus which infects almost all of the world's population subclinically during childhood and thereafter remains for a life. Immunocompromised persons often show active EBV infection, which may progress to virus-associated lymphoproliferative disorders. Many clinical researches show a strong role for viral load measurement in predicting and monitoring EBV-associated diseases, especially in immunosuppressed patients. The aim of this work was to design and to optimize novel real-time PCR assay for detection and quantification of Epstein-Barr virus DNA. METHODS In described experiment TaqMan chemistry-based primers and probes were designed to specific EBV sequence of BALF5 viral gene. To test laboratory utility of the designed method, 80 sera samples, positive for EBV DNA in routine investigations, were also analyzed and 1st International WHO WBV Standard was applied for recalculation of the results to international units. RESULTS Developed real-time PCR assay gave positive result only in the samples containing genetic material of EBV. Mean viral load of the 80 clinical samples tested was 2,838 and 3,241 log10 copies/ml for analyzed and reference method, respectively. Correction with EBV Standard led to equalization of these results (3,229 and 3,244 log10 international units/ml respectively). CONCLUSIONS The obtained data indicate that this TaqMan-based qPCR assay is accurate, rapid and reliable method for the diagnosis and monitoring of EBV DNAemia in clinical samples, coming from immunosuppressed individuals.