Objectives: This study aimed to determine the rate of unnecessary test requests in a university hospital biochemistry laboratory by evaluating tests against the Turkish Ministry of Health’s “Rational Test Request Procedure” guidelines, specifically focusing on “Test Request Duration (Day)”. Methods: This retrospective observational study analyzed five biochemistry tests (Vitamin B12, Vitamin D, LDL cholesterol, LDH, and folate) with the longest recommended retesting periods. The study covered 30 months (July 6, 2019 – January 7, 2022) to compare test request patterns before and after the national policy’s implementation. Data from the hospital information system defined unnecessary requests as those ordered for the same patient before the minimum suggested retesting interval. Associated costs were estimated using official Social Security Institution reimbursement values. SPSS v26 was used for frequency analyses and Chi-square tests to assess associations between unnecessary ordering and physician gender/title, patient age/gender, and clinical setting. Results: Of 490,344 test requests made, 199,553 were inappropriate (40.7 %), which accounted for 49.0 % cumulative spend. LDH had highest inappropriate use (61.7 %), with over 90 % of internal medicine test requests identified as redundant. Decreased considerably were test requests for Vitamin D by 70 % since policy introduction. Conclusions: In spite of national standards, improper test use endures when it comes to non-specific biomarkers. The dramatic overutilization of LDH was identified as the most significant cause of redundant testing and expense. Directed training as well as decision-support tools are thus imperative in order to streamline wasteful testing and downstream improvement.