HEV infections in animals are mainly diagnosed based on the presence of anti-HEV antibodies in sera using enzyme-linked immunosorbent assays (ELISA). In fact, commercial HEV ELISA kits are only available for selected animal species excluding rabbits. The demonstration of cross-reactivity of rabbit anti-HEV antibodies with human HEV strains makes possible diagnostic use of (human) ELISA kits for detection of specific anti-HEV antibodies in rabbit sera. The aim of the study was the application of ELISA recomWell HEV IgG (human) for identification of HEV seropositive rabbits at the time of slaughter. Native recombinant protein A was used as broadly reactive conjugate characterized by cross-species reactivity with IgG antibodies of human and other animal species. Several dilutions of protein A were employed and subsequently tested against the panel of control sera (positive, negative, and borderline) to determine its optimal concentration. Statistical analysis of OD values and their corresponding antibody titers obtained for each group of tested sera and protein A showed the most consistent performances at 1:40,000 dilution when compared to anti-human IgG (control) conjugate. Finally, the correct performance of the optimized test was verified using rabbit serum samples containing (or not) specific anti-HEV antibodies. As demonstrated here, HEV IgG (human) ELISA employing ORF2 antigens of human gt1 and gt3 HEV strains after modification and subsequent optimization can be used for the detection of anti-HEV antibodies in rabbit sera.