Purpose The reference range described in Adrenocorticotropic Hormone reagent used in our laboratory is 10-60 pg/mL at 8 a.m. to 10 a.m., and 6-30 pg/mL at 8 p.m. to 10 p.m. However, in the case of outpatients, blood is mainly collected between 10 a.m. and 6 p.m., accounting for 57.8% of the total. Therefore, This study is intended to help make a more accurate diagnosis by reevaluating the reference range provided by the manufacturer of the Adrenocorticotropic Hormone reagent and setting split-timed reference range. Materials and Methods The patients collected blood before 10 a.m. were group A (68 people), and the patients collected blood after 10 a.m. were set to group B (80 people). A T-test was performed between groups to test their significance. And it was confirmed whether it was necessary to set the gender classification as a subgroup. The method of setting the reference range was calculated by the Bayesian's method and the Hoffmann's method. Results The reference range of Group A was 8.6 to 60.6 pg/mL by the Bayesian's method, and the Hoffmann's method was 3.6 to 61.3 pg/mL. The reference range of Group B was 6.9 to 50.5 pg/mL when applying the Bayesian's method, and the Hoffmann method's was 2.3 to 48.9 pg/mL. Conclusion This study was concluded that it was necessary to set the split-timed reference range. Through this study, the later the blood collection time, the lower the level of Adrenocorticotropic Hormone, indicating that blood collection time is important for patients with clinical significance. If a large number of subjects are selected and supplemented in the future, it is believed that systematic and accurate reference range can be set.