Abstract Introduction/Objective With rising healthcare costs in the United States, there has been a push for lab stewardship to improve the quality of patient care while reducing costs. To optimize the use of clinical laboratory testing, the ASCP working with other medical specialty organizations, developed the Choosing Wisely Campaign to promulgate evidence-based guidelines to optimize clinical laboratory testing. Methods/Case Report We examined adherence to three Choosing Wisely guidelines over a four-year period (2017- 2020), through queries of internal cost accounting databases to return aggregate volumes as well as variable and total costs at three large academic health systems. We measured concurrent orders for: 1) erythrocyte sedimentation rate (ESR) with C-reactive protein (CRP), 2) serum/plasma amylase with lipase, and 3) free thyroxine (FT4) and/or total triiodothyronine (TT3) with thyroid stimulating hormone (TSH) when the TSH is within the reference range (using an frequency estimate of 85% based on other studies). We also examined another guideline for concurrent orders for serum aldolase with creatine kinase (CK). We also quantified aggregate variable costs for the non-recommended test in each Choosing Wisely guideline (amylase, ESR, FT4 and/or TT3), and for serum aldolase when ordered with CK. Results (if a Case Study enter NA) Over the four-year period, there were 322,853 unnecessary tests based on these four guidelines (120,587 ESR and CRP, 30,444 amylase and lipase, 164,818 FT4 and/or TT3 with TSH, and 7,004 aldolase). Overall, unnecessary testing decreased between 2017 and 2020 for amylase with lipase, remained essentially unchanged for aldolase, and increased for the other two test guideline scenarios. The largest changes were concurrent orders for amylase and lipase at one health system (38% decrease), and orders for TT3 with a normal TSH result at another health system (324% increase). The four-year variable cost of these unnecessary tests was $1,215,309 ($303,827 mean annual cost), resulting in potential annual variable cost savings of $101,276 for each health system for the four guidelines we examined. Variable costs for unnecessary testing increased by 16.5% ($45,571) over the four-year period. Conclusion Guideline-based unnecessary testing remains as a target to improve laboratory diagnostic testing. There is potential to realize significant achievable cost savings if guidelines are implemented and maintained.