Overestimation of immunoassays for cyclosporine (CsA) and tacrolimus (TAC) analysis in human whole blood is a problem. The liquid chromatography tandem mass spectrometry is recommended as a golden method for CsA and TAC analysis. The aim of the study is to develop and validate an ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method for simultaneous determination of CsA and TAC in human whole blood and evaluate its agreement with a chemiluminescence microparticle immunoassay (CMIA). The UHPLC-MS/MS method for simultaneous determination of CsA and TAC in human whole blood was developed and validated according to the guidelines. A total of 177 CsA and 220 TAC samples were determined by UHPLC-MS/MS and CMIA, and the agreement of the two methods was evaluated by Bland-Altman plot. The calibration range of UHPLC-MS/MS method was 5 to 2000 ng/mL for CsA and 0.2 to 80 ng/mL for TAC. The inaccuracy and imprecision were - 13.33% to 11.80% and < 11.74% for CsA and - 8.94% to 6.53% and < 10.84% for TAC, respectively. Evaluated by Bland-Altman plot, the mean overestimation of CMIA compared to UHPLC-MS/MS was 53.7% for CsA and 48.1% for TAC.