Abstract Objectives Autoantibodies and, specifically antinuclear antibodies (ANA), are the hallmark of systemic autoimmune diseases (AID). In the last decades, there has been great technical development to detect these autoantibodies along with an increased request for this test by clinicians, while the overall pre-test probability has decreased. In this study, we compare the diagnostic performance of three different methods for ANA screening (indirect immunofluorescence [IIF], addressable laser bead immunoassay [ALBIA], and fluorescence enzyme immunoassay [FEIA]). Methods Serum samples at baseline visit from 2,997 participants from the Camargo Cohort, a population with an overall low pre-test probability for systemic AID, were analyzed with the three methods. Participants have a minimum follow-up of 10 years and the development of autoimmune diseases was collected from clinical records. Results The highest frequency of positive ANA was observed by IIF assay. However, ALBIA showed high sensitivity for AID. Likewise, solid phase assays (SPA) presented higher specificity than IIF for AID. ANA prevalence with any method was significantly higher in females and overall increased with age. Triple positivity for ANA was significantly related to the presence of anti-dsDNA–SSA/Ro60, Ro52, SSB/La, RNP, Scl-70, and centromere–specificities. No association was found for anti-Sm – RNP68, or ribosomal P – specificities. Noteworthy, triple positivity for ANA screening was associated with diagnosis of systemic AID both at baseline visit and follow-up. Conclusions ANA detection by IIF may be better when the pre-test probability is high, whereas SPA techniques are more useful in populations with an overall low pre-test probability for systemic AID.