Antinuclear antibodies (ANA) are associated with connective tissue diseases (CTD). Indirect immunofluorescence (IIF) is the “gold standard” test but is time-consuming with high inter-observer variability. The objective is to compare IIF against the fully automated method, fluorescence enzyme immunoassay (FEIA) for initial screening for CTD. MEDLINE, EMBASE and Cochrane database searches (2000-March 2018) were conducted to identify fully-paired, cross-sectional or case-control studies of ANA screening for CTD reporting diagnostic results for FEIA and IIF. Study quality was assessed using the QUADAS-2 checklist. The reference standard was assessed against established classification criteria. The meta-analysis was conducted using hierarchical, bivariate, mixed-effect models. Ten studies were included in the meta-analysis. The model incorporating random-effects by test had the highest likelihood. IIF at a cut-off of 1:160 (7 studies, 3251 patients) or 1:80 (7 studies, 12311 patients) has a higher sensitivity compared to FEIA [1:160 0.86 (95% CI 0.75, 0.93) versus 0.74 (95% CI 0.65, 0.81); 1:80 0.89 (95% CI 0.84, 0.93) versus 0.78 (95% CI 0.71, 0.84)] but lower specificity [1:160 0.81 (95% CI 0.73, 0.87) versus 0.93 (0.91, 0.95); 1:80 0.72 (95% CI 0.62, 0.81) versus 0.94 (95% CI 0.90, 0.96)]. Based on a CTD prevalence of 2.7%, for every 1,000 screening tests, on average seven patients with CTD will have a false negative test with FEIA compared to four and three for IIF at 1:160 and 1:80. Furthermore, 64 patients without CTD will have a false positive test with FEIA compared to 184 and 269 for IIF at 1:160 and 1:80 respectively. Given the high specificity for CTD, FEIA may be valuable to screen for CTD alongside IIF. Clinicians should be acquainted with the clinical presentation of CTD and should be aware of the laboratory tests used and their advantages and disadvantages in order to avoid misdiagnosis.