Therapeutic drug monitoring is an essential tool when managing the therapeutic use of immunosuppressant cyclosporine A (CsA) in cases with solid organ transplantation. In China, the concentration of CsA is primarily measured using immunoassays. However, existing literature recommends mass spectrometry as the current gold standard for the quantitation of CsA. In the present study, it was attempted to develop a novel application to determine CsA concentrations by using ultra-performance liquid chromatography coupled to high-resolution mass spectrometry (UPLC-HRMS). This technique was then compared with a commercially available chemiluminescent microparticle immunoassay (CMIA) and it was investigated how clinical factors may contribute to quantitation differences between the two methods. An UPLC-Orbitrap-MS method was developed to determine CsA concentrations and this method was validated using guidelines put forward by the Food and Drug Administration from the US. In total, 127 blood samples were acquired from patients undergoing kidney transplantation and analyzed by UPLC-HRMS and CMIA assays. The novel method provided sensitive, accurate and precise results. The mean CsA concentration measured by CMIA was significantly higher than that measured by UPLC-HRMS (85.70 +/- 48.99 vs. 67.06 +/- 34.56 ng/ml, P<0.0001). Passing Bablok analysis yielded a slope of 1.34 (95% CI: 1.22-1.47) and an intercept of -2.54 (95% CI: -10.29-5.52). A group of samples with a higher metabolic ratio (hydroxylated CsA/CsA>1) exhibited larger discrepancies, while a group of samples taken from patients with a longer post-transplantation time (>10 years) featured narrow 95% CIs from -15.32 to 65.69%, as determined by Bland-Altman analysis. In summary, a reliable, accurate and rapid UPLC-HRMS method for CsA analysis was successfully developed. The measurement of CsA by the CMIA assay in renal transplant patients should be further evaluated with a specific focus on positive bias.