Objective: To determine the rates of simultaneous antinuclear antibodies (ANA) screening and extractable nuclear antigen (ENA) testing that do not follow recommendations.Design, Setting, and Participants: Retrospective cohort study of adult patients (≥18 years) with a HEp-2 ANA or ENA ordered in the Marshfield Clinic Health System.Main Outcome(s) and Measure(s): Counts of patients having simultaneous ANA and ENA laboratory testing or ENA testing without ANA screening. Relevant ENA positivity in ANA negative patients. Secondary measures included relative timing of ANA and ENA ordering, potential cost savings of unnecessary testing, and provider ordering characteristics including specialty and provider type.Results: Of 58,627 cohort patients, 39,155 (66.8%) were women, and the mean (SD) age at first laboratory testing was 48.7 (19.0) years. The negative ANA with positive ENA rate was 2%. Further stratification identified only 23 diagnosed autoimmune connective tissue diseases (AI-CTDs) in this 2%, with a resulting negative ANA with relevant positive ENA rate of 0.37%. Simultaneous ANA and ENA testing occurred in 8.3% of patients, and an ENA only was ordered in 24.2% of patients. The simultaneous or non-sequential ordering of ANA and ENA testing resulted in significant health care costs of $2,293,251.80 over 20,112 unique patients.Conclusions and Relevance: A significant percentage of providers do not follow recommendations to sequentially order ANA and ENA testing on patients with suspected AI-CTDs. Significant saving in health care spending without failure to diagnose AI-CTDs can be achieved if ANA testing is performed first, followed by ENA testing when suspecting AI-CTDs in patients.