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Herpes simplex virus type 1 (HSV1), establishes life-long latency and can cause symptoms during both first-time infection and later reactivation. The aim of the present study was to describe a protocol to generate a reliable and discriminative avidity index (AI) for anti-HSV1 IgG content in human sera. Human serum from two distinct cohorts; one a biobank collection (Betula) (n = 28), and one from a clinical diagnostics laboratory at Northern Sweden University Hospital (NUS) (n = 18), were assessed for presence of IgG antibodies against HSV1 by a commercially available ELISA-kit. Addition of urea at the incubation step reduces effective binding, and the ratio between urea treated sample and non-treated sample was used to express an avidity index (AI) for individual samples. AI score ranged between 43.2 and 73.4% among anti-HSV1 positive biobank sera. Clinical samples ranged between 36.3 and 74.9%. Reproducibility expressed as an intraclass correlation coefficient (ICC) was estimated at 0.948 (95% CI: 0.900-0.979) and 0.989 (95% CI 0.969-0.996) in the biobank and clinical samples, respectively. The method allows for AI scoring of anti-HSV1 IgG from individual human sera with a single measurement. The least significant change between two measurements at the p < 0.05 level was estimated at 5.4 and 3.2 points, respectively, for the two assessed cohorts.