To determine the seroprevalence of HBoV1, 2, 3, and 4 in humans, we developed an ELISA protocol for detecting IgG antibodies against HBoV1-4 using VLPs as coating antigens. The concentration of the coated VLPs (0.125 mg/mL) for this ELISA assay was optimized using chessboard titration tests. The positive sera and negative sera showed different results at 1:200 serum dilutions. As the results obtained using lower serum dilutions were similar to those obtained at 1:200 (data not shown), the 1:200 dilution was used in subsequent ELISA analysis of serum samples.