Aim. To determine the threshold value of the Epstein-Barr virus (EBV) viral load (VL) in blood leukocytes to improve the laboratory diagnostics of infectious mononucleosis in children.Materials and methods. EBV DNA quantification in blood leukocytes in children aged 1-17 years (n=163) was determined by real-time polymerase chain reaction. VL were compared in groups of EBV mononucleosis (n=67), non-EBV mononucleosis (n=25) and healthy donors (n=25). Threshold was determined based on VL data from children with active and latent EBV infection. The R program and the RStudio environment were used for satistic analysis.Results. EBV DNA is found in blood leukocytes in infectious mononucleosis not associated with EBV and in healthy virus carriers, however VL in these groups is significantly lower than in patients with EBV mononucleosis (p<0.001). The threshold value was determined – 41 copies/105 cells (or 1.6 lg of EBV DNA/105 cells), which was characterized by acceptable values of specificity and sensitivity (0.90 and 0.85, respectively) of laboratory diagnostics. High EBV VL (equal to or above the set threshold) is associated with an 8.5-fold increased risk of detecting active EBV infection compared to children who have a low VL (below a set threshold) (RR 8.5; 95% CI: 3.7–19.7, p<0.001).Conclusion. In general, the results obtained create prerequisites for more intensive implementation of quantitative studies of EBV DNA in blood leukocytes, both in the context of improving the early diagnosis of infectious mononucleosis and its etiological interpretation, and in terms of detailing the features of the course of EBV infection.