Insulin degrading enzyme (IDE) plays a critical role in degrading insulin and beta-forming proteins, implicating its significance as a biomarker in metabolic dysfunction and neurocognitive disorders, including Alzheimer's disease (AD). Understanding the impact of pre-analytic conditions of in vitro IDE levels is imperative for reliable biomarker assessment. This study explored the influence of freeze-thaw cycles, storage temperature, and storage time on IDE levels in human serum. Serum samples from seven healthy volunteers were subjected to various storage conditions, including refrigeration (4 °C) and freezing (-20 °C and -80 °C) for 24 h and six months, with differing freeze-thaw cycles. In vitro IDE levels were measured at 24 h and after 6 months using ELISA. Results indicate that while short-term storage at either -20 °C or -80 °C yielded similar IDE levels, prolonged storage and multiple freeze-thaw cycles significantly impacted IDE stability, with colder temperatures exhibiting better preservation. Although further research with larger cohorts and longer storage time is warranted to establish clinical significance, our study suggests preferential use of unthawed samples or consistent freeze-thaw conditions for accurate IDE assessment. Thus, optimizing sample storage conditions is paramount for reliable IDE biomarker analysis in clinical and research settings.