Background: Antinuclear antibodies (ANAs) are useful for the diagnosis of ANA-associated systemic rheumatic disease (AASRD). The objective of this study was the evaluation of an immunoassay that detects antibodies to a mixture of 17 antigens as an alternative to indirect immunofluorescence (IIF). Methods: Nine thousand eight hundred and fifty-six consecutive patients tested for ANAs were tested by IIF and EliA connective tissue disease screen (Thermo-Fisher). Medical records were reviewed for 2475 patients, including all patients that tested positive/equivocal by either test and a selection of 500 patients that tested negative. Results: Concordance between IIF and EliA was 83.1%. AASRD was found in 12.8% of IIF-positive patients, 30.2% of EliA-positive patients and 0.4%, 46.6%, 5.8% and 3.0% of patients that tested, respectively, double negative, double positive, single positive for EliA and single positive for IIF. The association with AASRD increased with increasing antibody level. IIF and EliA were positive in, respectively, 90.4% and 69.9% of systemic lupus erythematosus (n=83), 100% and 84.1% of systemic sclerosis (n=63), 86.7% and 93.3% of Sjögren’s syndrome (n=45), 88.2% and 52.9% of polymyositis/dermatomyositis (n=17), and in all cases of mixed connective tissue disease (n=8). The specificity was projected to be 94%–96% for EliA and 86% for IIF. When all AASRDs were taken together, the areas under the curve of receiver operator curves were similar between IIF and EliA. Conclusions: The positive predictive value for AASRD was higher for EliA than for IIF, but, depending on the disease, EliA might fail to detect antibodies that are detected by IIF. Combining immunoassay with IIF adds value.