This study investigated the stress-related metabolites and hormones in blood and compared the muscle structure to identify the reason for blood splash in Hanwoo beef. Five slaughter houses were selected based on the region (Seoul, Gimhae, Jungbu, Naju, and Goryung) and a previous blood splash record. In total, three-hundred eighty blood samples (n=380) and forty-two muscle tissues (n=42) of control and blood splash Hanwoo beef were collected during the slaughter process and beef grading. Blood metabolites were analyzed including glucose, lactate, creatinine, urea-N, and hormones such as cortisol and thyroxin. Muscle fiber, fiber bundle, and capillary wall thickness were measured under microscope. The concentrations of blood glucose, lactate, and urea-N were not significantly different between the control and the blood splash samples. Cortisol and thyroxin levels were not significantly different in both samples. In contrast, the creatinine level was significantly increased (p<0.05) in the blood splash samples. There were also no significant differences observed in muscle fiber, bundles, and capillary wall thickness between the control and the blood splash tissues. In conclusion, blood metabolites, hormones, and muscle fiber showed no differences between the control and the blood splash animal. However, increased creatinine levels may be used as an indicator for identifying blood splash prior to slaughter in Hanwoo.