Erythropoietin-producing hepatocellular receptor A8 (EphA8) is a type I transmembrane protein that belongs to the largest Eph family among receptor tyrosine kinases. By binding to its membrane-bound ephrin-A or ephrin-B ligands on adjacent cells, Eph receptors form complexes and mediate bidirectional signaling activities, triggering cell-cell adhesion and repulsion. Increased expression of EphA8 correlates with poor prognosis in some types of cancer. Therefore, developing sensitive monoclonal antibodies (mAbs) for EphA8 has been desired for treatment, diagnosis, and further basic research. In particular, there are no anti-EphA8 mAbs that can be used for flow cytometry. In this study, we established a novel specific and sensitive anti-human EphA8 mAb, which applies to flow cytometry, clone Ea8Mab-9 (mouse IgG1, kappa), by the Cell-Based Immunization and Screening (CBIS) method. Ea8Mab-9 reacted with EphA8-overexpressed Chinese hamster ovary-K1 cells (CHO/EphA8) and EphA8-overexpressed LN229 glioblastoma cells (LN229/EphA8) in flow cytometry. Ea8Mab-9 did not recognize other members of Eph receptors. Furthermore, Ea8Mab-9demonstrated a high binding affinity for CHO/EphA8 and LN229/EphA8 with dissociation constants of 1.3×10-9 M and 1.6×10-9 M, respectively. Ea8Mab-9, established by the CBIS method, could be useful for analyzing the EphA8-related biological response using flow cytometry with high affinity and specificity.