OBJECTIVE: To understand the ordering patterns of the antinuclear antibody (ANA) multiplex testing in a single, large Department of Defense (DoD) tertiary healthcare system. METHODS: Records of patients with an ANA multiplex assay ordered over a one-year period were evaluated in a large DoD hospital system. Duplicate tests and patients with a previously established Autoimmune Rheumatic Disease (ARD) prior to the year of study were excluded. The remaining 2,499 patients' charts were reviewed for clinical presentation, ordering specialty, ordering rationale, and whether subsequent rheumatology evaluations resulted in a new ARD diagnosis. RESULTS: The ANA multiplex assay was ordered most often by primary care and medicine subspecialties for over 100 reasons. In the ANA multiplex assay negative group n = 37/2,228,1.66% individuals were diagnosed with a new ARD. In the ANA multiplex assay positive group n = 37/271, 13.7% individuals were diagnosed with a new ARD. Sjögren disease, systemic lupus erythematosus, and undifferentiated connective tissue disease were the most common newly diagnosed ARDs in the ANA multiplex assay positive group. Rheumatoid arthritis and seronegative spondyloarthritis were the most common new ARD diagnoses in the ANA multiplex assay negative group. In this study, 97% of the ordered ANA assays did not lead to an ARD diagnosis. CONCLUSION: This study demonstrates frequent utilization of the ANA multiplex assay in the evaluation of non-specific signs and symptoms with a low rate of ANA associated ARDs suggesting a need for implementation of strategies to improve understanding of appropriate clinical contexts that warrant ANA testing.