Autoantibodies against centromere protein ‐F have been reported to be associated with various types of cancer with poor prognosis. The characterization of these autoantibody specificities is important in both diagnostics and basic research. In this study, we mapped the epitope (NELSRIRSEKA) of two monoclonal centromere protein F antibodies. The epitope was localized by screening of overlapping peptides followed by a fast and efficient estimation of the minimal peptide length required for antibody recognition, based on the screening of terminally truncated resin‐bound peptide analogs. The epitope was determined through competitive inhibition assays of systematically truncated free peptides. In addition, the importance of the involved amino acid side chains of the identified epitope was determined through competitive inhibition assays using alanine‐substituted analogs.