Mice have been increasingly used in pharmacokinetic evaluation due to using less amount of compound and availability of various disease models and blood sampling methods in mice have produced the distinct pharmacokinetic profiles even in the same compounds. In this study, three blood sampling methods [i.e., automated dosing/blood sampling (ABS), tail artery collection (TA) and heart puncture (HP)] were applied to investigate the pharmacokinetics of moracin C in mice. Depending on blood sampling sites, significant differences were observed in plasma concentrations as well as pharmacokinetic parameters such as AUC (area under the plasma‐time concentration curve). The AUC values in ABS and TA groups were smaller than that in HP group. Considering that moracin C was greatly distributed to liver and gastrointestinal tracts (e.g., stomach, small intestine, and large intestine) and was extensively metabolized via glucuronidation in liver and small intestines, the AUC differences could be mainly caused by the stronger stress from blood sampling via heart puncture, which might interfere the elimination (i.e., hepatic and/or intestinal first‐pass effects). The remaining amounts of moracin C in gastrointestinal tract at 24 h after its oral administration were similar among three groups with different blood sampling methods, indicating that the absorption of moracin C was not affected during the blood sampling stress of HP. These results indicate that the ABS or TA method can be utilized to obtain better reliable and accurate pharmacokinetic parameters in mice.Support or Funding InformationThis study was supported by National Research Foundation of Korea (NRF) grants funded by the Korea government (MSIT) (NRF‐2016R1C1B2010849, NRF‐2019R1A2C2009053 and NRF‐2018R15A2023217).