Detection of hepatitis B virus (HBV) serological markers in dried blood spot (DBS) samples by enzyme immunoassay (ELISA) has not yet been fully optimized. In this study, the ability to detect three HBV markers (HBsAg, anti-HBc, and anti-HBs) was evaluated in DBS samples using a modified commercial ELISA. Matched serum and DBS samples were obtained from individuals with or without a past history of HBV infection. Sera samples were tested according to the manufacturer's instructions, but for DBS testing, paper diameters, elution buffer, volume of input sample, and cut-off values were evaluated to optimize the assay. Stability studies were done on DBS stored at for up to 180 days at different temperatures. The absorbance values that yielded the maximum sensitivity and specificity were determined based on the area under the ROC curve (AUROC) and chosen as the cut-off value. Using this parameter, sensitivity was 90.5%, 97.6%, and 78% for anti-HBc, HBsAg, anti-HBs assays, respectively. Specificity was 92.6%, 96.7%, and 97.3% for anti-HBc, HBsAg, and anti-HBs assays, respectively. HBV markers could be detected in DBS samples until 63 days after sample collection at most temperatures, but storage at -20°C yielded more consistent results. These results indicate that modified ELISA can be used to detect HBV markers in DBS samples, particularly if the samples are stored appropriately.