Abstract: In the present study an indirect in-house ELISA was optimized to detect IgG antibody against HHV-6 in sera from different groups of children. Regarding designation of in-house ELISA, all corresponding steps were standardized by checker boading after virus culture and purification of antigen. The results were calculated according to Specific Binding Ratio (SBR) and cut-off methods. Inter and intra assay helped to investigate different technical aspects of in-house ELISA. When accuracy of ELISA was confirmed the protocol was applied in screening of sera from different hosts including immunocompetent and immunocompromized that facilitated examination of their clinical aspects. Furthermore, seropositivity and seronegativity had to be proved real or false so Diethylamine (DEA) in 20 and 70 mM concentrations were used in order to assay IgG antibody avidity. Primary HHV-6 infection and seroconversion are reported most commonly between 6 and 18 months of age and among children older than 3 years most subjects are seropositive. Nonetheless, a high prevalence of HHV-6 IgG antibodies was documented in all children if all SBR and/or cut-off positive HHV-6 IgG ELISA readings were considered to represent seropositivity. No evidence of a decline in HHV-6 antibody positivity was seen with increasing age amongst children. The HHV-6 IgG antibody avidity indices were stable throughout childhood in immunocompromized patients. The in-house HHV-6 ELISA proved efficient, reproducible and quite concordant with results obtained by previous workers in terms of IgG distribution of the virus.