Overlapping and non-specific clinical signs are the hallmark of autoimmune diseases. This may lead to delays in diagnosis and appropriate treatment. To facilitate appropriate referral to specialists, laboratory testing should be considered for patients who manifest symptoms consistent with one or more autoimmune diseases. The antinuclear antibody (ANA) immunofluorescence assay (IFA) is a first-line screening test for patients with a suspected autoimmune disease. This test is the gold standard because of its high sensitivity compared to other assays. ANA IFA detects up to 150 antibodies and if positive, reflexes to titer and pattern, which help provide insight as to which autoimmune disease may be present. Higher titers are generally associated with greater likelihood of rheumatic disease. Patterns are associated with various autoimmune diseases. Positive ANA IFA results should prompt clinicians to continue investigating the cause of a positive ANA IFA and narrow the field of potential suspects. The American Autoimmune Related Diseases Association reports it takes on average 4.6 years and nearly 5 doctor visits to receive a proper autoimmune disease diagnosis. Laboratory testing correlated with clinical finding is key to differential diagnosis, disease classification and patient care. This lecture describes how the ANA IFA screen in combination with reflex testing to specific autoantibody tests can be used effectively for differential diagnosis with one blood draw for the patient. It further describes titer and patterns associated with ANA IFA testing and presents 3 case studies for learning purposes.