Antinuclear antibodies (ANA) are a group of autoantibodies directed against nucleic cellular antigens of the organism itself, as well as against other cell factors (nucleic and cytoplasmic) . Positive ANA profile is mostly found in connective tissue disorders (CTDs), such as systemic lupus erythematosus (SLE), Sjögren’s syndrome (SS), rheumatoid arthritis (RA), systemic sclerosis, mixed connective tissue disease, and polymyositis/dermatomyositis. Their role in the pathogenesis of these diseases has been a subject of research, but the question of whether they are indeed the cause or the result of the autoimmune response remains unanswered. They are recognized as important diagnostic and prognostic markers for CTDs and their detection is used as a routine test for the evaluation of a suspected autoimmune disorder . It is known that these antibodies can also be detected in patients suffering from non-autoimmune diseases, such as infectious diseases, allergies, or malignancies [2, 3]. ANA are also detected, in low frequencies, in apparently healthy individuals. The frequency of ANA in the healthy population ranges from 1.1–31.7 % [4–10]. This wide range of frequencies is due to a variety of factors, such as age, race, use of drugs, exposure to xenobiotics, and the laboratory method used for their detection . For decades, indirect immunofluorescence assay (IFA) has been used as the gold standard method for ANA detection. However, nowadays, new automated enzyme-linked immunosorbent assay (ELISA) methods are increasingly being adopted as a routine method for ANA screening . The purpose of this study was to determine the presence of ANA in healthy individuals using IFA, as well as an enzyme-linked immunoassay (Hep-2 whole cell as antigen), in relation to age and gender. All positive ANAwere further examined for specific autoantibodies in order to evaluate the clinical significance of positive ANA in healthy individuals.