Introduction: The term anti-nuclear antibody (ANA) is used to define a large group of autoantibodies which specifically bind to nuclear elements. Although healthy individuals may also have ANA positivity, the measurement of ANA is generally used in the diagnosis of autoimmune disorders. However, various studies have shown that ANA testing may be overused, especially in pediatrics clinics. Our aim was to investigate the reasons for antinuclear antibody (ANA) testing in the general pediatrics and pediatric rheumatology clinics of our hospital and to determine whether ANA testing was ordered appropriately by evaluating chief complaints and the ultimate diagnoses of these cases. Methods: The medical records of pediatric patients in whom ANA testing was performed between January 2014 and June 2016 were retrospectively evaluated. Subjects were grouped according to the indication for ANA testing and ANA titers. Results: ANA tests were ordered in a total of 409 patients during the study period, with 113 positive ANA results. The ANA test was ordered mostly due to joint pain (50% of the study population). There was an increased likelihood of autoimmune rheumatic diseases (ARDs) with higher ANA titer. The positive predictive value of an ANA test was 16% for any connective tissue disease and 13% for lupus in the pediatric setting. Conclusion: in the current study, more than one-fourth of the subjects were found to have ANA positivity, while only 15% were ultimately diagnosed with ARDs. Our findings underline the importance of an increased awareness of correct indications for ANA testing.