Abstract: LC-MS/MS method monitoring tacrolimus (FK506) in human whole blood for therapeutic drug monitoring(TDM) was developed. FK506 and the internal standard were extracted from whole blood by acetonitrile which used as deproteinated solvent, and separated on a Shim-pack VP-ODS column using methanol and 10 mmol•L-¹ammonium acetate as mobile phase by gradient elution. Detection was carried out by multiple reaction monitoring on a 3200QTRAP LC-MS/MS system. The assay is linear over the range 0.5-50.0 μg•L^-1with a limit of quantity of 0.5 μg•L^-1 and a limit of detection of 0.1 μg•L^-1. Intra-day and inter-day precision are less than 6.8%, respectively. The relative deviation is in the range of -2.99% to 2.23%. The recoveries of FK506 are nearly 100.0%, and stabilities are good. The method is a rapid, sensitive, selective and reliable method for the determination of monitoring FK506 for TDM.