Glucose is a major circulating carbohydrate in birds and its level in the blood is often used as a biometric indicator in clinical diagnosis and various studies. Notably, hypoglycemia is often associated with Spiking Mortality Syndrome in broilers; therefore, blood glucose levels need to be correctly evaluated in clinical diagnosis. In the present study, we investigated the effect of different blood treatment methods after blood collection on chicken blood glucose measurements. The blood glucose level of plasma separated from blood cell components immediately after blood collection was used as a reference and compared with glucose levels in serum and stored plasma. The mean glucose level in plasma separated from blood cell components immediately after blood collection was 236.1±15.9 mg/dL and remained stable for at least one week in refrigerated storage (between 2°C and 5°C). However, glucose levels decreased slowly in plasma unseparated from blood cell components in storage with ice water. Mean glucose level in serum separated from blood cell components 1 h after blood collection was 206.4±9.2 mg/dL and fell to 108.3±30.0 mg/dL after 24 h. Therefore, the chicken blood serum glucose level was significantly lower than the level in plasma immediately after blood collection, regardless of elapsed time after blood collection. For the measurement of glucose in chicken blood, it is necessary to use refrigeration, use plasma from which blood cell components have been removed, and take measurements within at least 30 min.