ObjectiveTo develop and validate an LC‐M/SMS method for the determination of tacrolimus in human whole blood.MethodThe LC–MS/MS method for the determination of tacrolimus in whole blood was developed and validated according to the guidelines. Concentrations of TAC in 100 kidney transplant patients measured by LC–MS/MS were compared with CMIA using correlation analysis and Bland–Altman plots.ResultsThe method had a total chromatographic run time of 5 min. The calibration curves were linear over the range of 0.5–100.0 ng/mL with a lower limit of quantification of 1 ng/mL. The intra‐ and interday accuracy was within the range of 93.3%–109.2% and 96.0%–108.4%, respectively, with precision ranging from 0.8 to 9.4%. The mean extraction recoveries of TAC ranged from 102.6 to 107.8%. The mean concentrations of TAC in whole blood of kidney transplant patients measured by the two assays were different at 1, 3 months and all time points (p < 0.001), but no significant difference was observed at 6 months (p = 0.094). The correlation of data was good with the correlation coefficients (r2) of 0.7581, 0.8811, 0.8777, and 0.8077, respectively. Passing–Bablok regression analysis demonstrated good correlations with r2 values higher than 0.88 between TAC levels measured by LC–MS/MS and CMIA. Using Bland–Altman plots yielded average biases of 1.29, 0.79, 0.11, and 0.65 ng/mL at 1, 3, and 6 months and all time points.ConclusionThe LC–MS/MS method was validated for the accurate determination of TAC in human whole blood. The comparison of tacrolimus concentrations measured by the LC–MS/MS with CMIA showed a good correlation and agreement of two methods, suggesting LC–MS/MS should be used routinely to monitor TAC concentrations in kidney transplant patients.