Antinuclear antibodies are autoantibodies targeting nuclear antigens. They are key players in diagnosing autoimmune diseases, the most prevalent being systemic lupus erythematosus, Sjögren’s syndrome and systemic sclerosis. However, their high prevalence in the general population complicates interpretation, as they can be positive, especially in low titres, even in healthy individuals and the elderly. Furthermore, as they are not specific to a disease, positive antinuclear antibodies may also be found in other autoimmune diseases such as Hashimoto’s thyroiditis, autoimmune hepatitis and also in chronic infections, malignancies or as a drug-induced phaenomenon. On the other hand, negative antinuclear antibodies do not rule out autoimmune diseases. For example, polymyositis and ankylosing spondylitis have negative antinuclear antibodies, Misinterpretation of these autoantibodies may lead to diagnostic faults. In addition, the titre is not correlated with disease activity, adding another diagnostic challenge. Advances in diagnostic criteria and the inclusion of more specific markers, such as anti-dsDNA or anti-Scl-70 antibodies, have improved diagnostic accuracy. For these reasons, clinicians must integrate antinuclear antibody testing with clinical findings in order to reach a correct diagnosis.