The aim of this study was to evaluate a commercially available ELISA assay for anti-nuclear antibodies (ANA) screening in a large routine laboratory setting. The detection of ANA is a commonly requested test by clinicians for patients suspected of rheumatic disease and other connective tissue diseases. Detection is part of the diagnostic criteria of rheumatic diseases such as SLE and can be important for monitoring purposes. ANA screening assays are typically indirect immunofluorescence (IIF), either on rodent tissue or HEp-2 cells; however, these are slow, subjective and laborious. In this study, in a routine serology laboratory setting, 2000 consecutive sera with requests for an ANA screen were tested by ELISA and results compared to those obtained by immunofluorescence. From these results we established an ANA ratio cut-off protocol to guide further action, a second series of 7000 samples was studied to assess the efficacy of this. Results show that the ANA ELISA can successfully replace IIF for the detection of clinically significant antibodies.