Objective To establish a micro volume blood sampling method via the saphenous vein for pharmacokinetic studies (PK studies) in mice, aiming at reducing animal use and alleviate animal pain in in vivo procedures. Methods CD-1 mice were intravenously or orally administered with model compounds A,B, C and D. Blood samples were collected by both the micro-sampling method and regular method at the same time points,and used for the measurement of plasma drug concentration. Pharmacokinetic parameters obtained from each method were compared. Results For each of the four compounds, plasma PK profiles generated by micro-sampling via saphenous vein were in good accordance with those by the regular sampling method via retro-orbital venous plexus. Conclusions Our newly developed micro-sampling blood collecting method can replace the regular blood collecting method used in pharmacokinetic studies in mice. It can significantly improve animal welfare by alleviating animal pain. The volume of blood withdrawn can be reduced by 80%, and the number of mice used for the pharmacokinetic studies can be reduced by 65%.