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Dried Blood Spots (DBS) are blood collection carriers that facilitate the storage and transport of samples. Used for quality control during sero-epidemiological investigations, DBS eluate are not the conventional specimen indicated by manufacturers for enzyme immunoassay method (EIA) for hepatitis B virus surface (HBs antigen). The aim of our study was to evaluate DBS eluates used as a matrix for EIA of HBs antigen in a reference laboratory. This study took place from August 2016 to November 2017 at the Centre for Diagnosis and Research on AIDS and other infectious diseases (CeDReS) in Abidjan, Côte d'Ivoire. We used a panel of 149 whole blood samples from blood donors. The DBS performed with these samples were analyzed after elution with the HBsAg (version ULTRA) ELISA, Dia.Pro Diagnostic Bioprobes S.R.L., Sesto San Giovanni, Italy. The technical performance (sensitivity and specificity and kappa coefficient) of the test performed on DBS was determined for different ratios (optical density/threshold value) compared to the results obtained on the plasma used as reference. We obtained a sensitivity of 100% with DBS for all ratios. The specificity increased according to the ratio of optical density of the individual EIA reaction to the threshold value, with 6.09%, 47.0%, and 83.0%, respectively, for ratios of 1.0, 3.0, and 5.0. Best performance was observed at ratio of 10.0 with a sensitivity and specificity of 100%. In conclusion, DBS eluate can be used for the diagnosis of viral hepatitis B and would be useful for conducting sero-epidemiological investigations. However, ratio giving best performance must be determined for each enzyme immunoassay method kits.