Detection of antinuclear antibodies (ANA) is a fundamental laboratory test for diagnosing systemic autoimmune diseases. Currently, the method of choice is indirect immunofluorescence (IIF) on a HEp-2 cell substrate. The goal of this study was to evaluate the diagnostic accuracy of five commercially available enzyme immunoassay (EIA) kits for ANA detection and to verify the possibility of using them as an alternative to the IIF method. The study involved 1513 patients, 315 of whom were diagnosed with a systemic autoimmune disease and 1198 in whom an autoimmune disorder was excluded. For all sera, ANA detection was performed via IIF and with five different EIA kits. The results were evaluated in relation to clinical diagnosis and the presence of possible specific autoantibodies (anti-ENA or anti-dsDNA); lastly, they were compared with the results obtained using ANA-IIF as the method of reference. The positive rate of the ANA-IIF test in subjects with systemic autoimmune diseases was 92%, whereas in the five ANA-EIA kits there was broad diversity in terms of response, with positive rates ranging from 74 to 94%. All the EIA kits correctly detected the presence of antibodies (anti-dsDNA, anti-RNP, anti-Ro/SSA) responsible for homogeneous and speckled fluorescence pattern, but at the same time they showed substantial inaccuracy with the nucleolar pattern, with a mean sensitivity of approximately 50% in this case. Instead, there was a large kit-to-kit difference in terms of identification of anti-Scl70 and centromere patterns, for which sensitivities ranged between 45 and 91%, and between 49 and 100%, respectively. The results of the study demonstrate that the commercially available ANA-EIA kits show different levels of sensitivity and specificity. Some of them have a diagnostic accuracy that is comparable and, in some cases, even higher than the IIF method. Consequently, these could be used as an alternative screening test to IIE. However, others do not ensure acceptable results. Therefore, careful evaluation of the various kits on the market is advisable before including any of these methods in the clinical and diagnostic testing.