Background: Antinuclear antibodies (ANA) are the serological hallmark of connective tissue diseases (CTD) and indirect immunofluorescence (IIF) on Hep-2 cells is still considered the gold standard for ANA screening. While this method is sensitive it lacks specificity. Moreover, low-titer ANA subspecificities may escape detection by IIF. Objectives: To investigate the usefulness of an ANA screening assay containing most of the diagnostically relevant antigens for CTD diagnostics. Methods: Sera from 265 consecutive patients presenting with symptoms characteristic of connective tissue diseases (but without a clear diagnosis yet) were analysed by IIF and the EliA ® CTD Screen (Thermo Fisher Scientific) containing the following antigens: dsDNA, U1-snRNP, Sm, Ro60, Ro52, La, Rib-P, topoisomerase I (Scl-70), centromere B, RNA polymerase III, fibrillarin, Jo-1, Mi-2, Pm/Scl. All positive sera were further analyzed by monospecific assays (Thermo Fisher Scientific). Results: Among the 265 patients, 90 were positive by IIF and 78 by CTD Screen; 61 sera were positive in both systems, 17 only in the CTD Screen and 29 only in IIF. In all double positive patients at least one diagnostically relevant antibody was detected, with anti-Ro and anti-dsDNA antibodies being most frequently detected. Importantly, antibodies were also detected in 15 of the 17 patients who were exclusively positive in the CTD Screen: 7 patients had anti-dsDNA, 4 anti-Ro, 1 anti-La, 2 anti-U1snRNP, and 1 patient had anti-Jo-1 antibodies. In contrast, among the 29 sera exclusively positive by IIF only two contained a diagnostically relevant antibody. Clinical evaluation revealed that 16 out of the 17 CTD Screen pos/IIF negative patients presented with at least 1 clinical sign commonly associated with systemic rheumatic disease (sicca syndrome, 12 patients; arthritis/arthralgias, 13 patients; microangiopathy, 2 patients; myositis, 2 patients; leukocytopenia, 2 patients; Raynaud's phenomenon, 5 patients; pericarditis, 1 patient; thromboembolic events, 2 patients). These patients may be at higher risk for developing a CTD, or, alternatively, may be at an early stage of a CTD in which a definite diagnosis is not yet to be made. The combination of distinct autoantibodies with clinical signs of systemic rheumatic disease, however, warrants a careful follow up in these patients. Conclusions: ANA screening assays containing multiple antigens such as the EliA ® CTD Screen seem to be helpful diagnostic tools that should be used in addition to IIF for detection of disease-associated autoantibodies enabling the physician to substantially improve diagnostics of connective tissue diseases.