Background: Various immunoassays have been used for cyclosporine A (CsA) analysis in human whole blood; however, they could not fully satisfy the requirements of criteria for accuracy and specificity in CsA measurement. The liquid chromatography tandem mass spectrometry is a gold method for CsA analysis. The aim of the study was to develop and validate an ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method for CsA analysis and establish its agreement with an antibody-conjugated magnetic immunoassay (ACMIA) in clinical sample analysis. Methods: An UHPLC-MS/MS method for CsA analysis in human whole blood was developed, validated, and applied in 85 samples, which were also tested by ACMIA. The agreement between UHPLC-MS/MS and ACMIA was evaluated by Bland-Altman plot. Results: The calibration range was 5-2000 ng/mL. The inaccuracy and imprecision were 24.60% to 5.56% and less than 8.57%, respectively. The internal standard-normalized recovery and matrix factor were 100.4%-110.5% and 93.5%-107.6%, respectively. The measurements of ACMIA and UHPLC-MS/MS were strongly correlated (r > 0.98). Evaluated by Bland-Altman plot, the 95% limit of agreement of the ACMIA: UHPLC-MS/MS ratio was 88.7%-165.6%, and the mean bias of the ratio was 21.1%. Conclusions: A rapid, simple, accurate, and reliable UHPLC-MS/MS method for CsA analysis in human whole blood was developed, validated, and applied in 85 samples. On average, 21.1% overestimation was observed in ACMIA compared with that in the UHPLC-MS/MS. Further and larger studies are required to identify whether this degree of variance could be accepted by clinicians.