Purpose: Insulin assays are highly sensitive to pre-analytical handling issues. While numerous studies have reported that the stability of insulin in non-hemolyzed samples is affected by time and temperature, there is limited information regarding the impact of delayed sample separation. Although research on this topic using the electrochemiluminescence immunoassay (ECLIA) method is actively conducted both domestically and internationally, studies utilizing the immunoradiometric assay (IRMA) are primarily performed abroad. Therefore, this study aimed to evaluate the stability of insulin in response to delayed separation of primary samples and storage conditions of secondary samples using the IRMA method. Subjects and Methods: The study included a total of 21 participants. Blood samples were processed and stored under different conditions to create serum and plasma samples divided into three groups. Group A: Secondary samples were stored at room temperature for 5 hours and at 2–8 °C for 48 hours. Group B: Secondary samples were stored at 2–8 °C for 5 hours and at -24 °C for 48 hours. Group C: Primary samples were stored at room temperature for 5 hours and at 2–8 °C for 48 hours before separation. Insulin concentrations in each group were measured using the IRMA method. The measured values were compared to insulin concentrations from secondary samples separated immediately after blood collection. The stability of insulin concentrations in each group was evaluated using regression analysis and the Wilcoxon signed-rank test for non-parametric paired sample comparisons. Statistical analysis was performed with MedCalc software (ver. 22.063), and a p-value of <0.05 was considered statistically significant. Results: The serum samples separated after storing the primary samples at 2–8 °C for 48 hours showed a positive correlation coefficient of 0.9974. However, the percentage deviation was -19.35%, and the p-value was <0.05, indicating a statistically significant difference. Similarly, for plasma samples stored as secondary samples at 2–8 °C for 5 hours and 48 hours, the correlation coefficients were 0.9803 and 0.9944, respectively, showing positive correlations. However, the percentage deviations were -7.45% and -6.32%, with p-values of <0.05 for both, demonstrating statistical significance. Conclusion: This study evaluated the stability of insulin assays under various conditions of delayed sample separation and storage methods. It is recommended that insulin assays be performed immediately after blood collection by promptly centrifuging and separating the samples. When storage is necessary, it is crucial to store samples in a separated state. Serum samples were stable when stored at room temperature or at 2–8 °C for 5 hours, and stability was maintained even after 48 hours of storage at 2–8 °C or -24 °C. However, delayed sample separation may result in significant differences in test values, emphasizing the need for careful handling and storage of samples.