Background Sirolimus, an immunosuppressive agent, is undergoing clinical trials in the prophylaxis of organ rejection. Objectives The aim of this study was to compare the performance of the semi-automated prototype (mode 1 A) microparticle enzyme immunoassay (MEIA) against a validated high-performance liquid chromatography-mass spectrometry (HPLC-MS) method for measuring sirolimus concentrations. A secondary objective was to identify potential factors that may influence sirolimus measurement. Methods The comparison was based on predose samples (n = 841) from 74 renal transplant patients receiving sirolimus therapy. Samples were collected up to 12 months after transplantation. Results The mean (± SD) overestimation by MEIA was 42.5% ± 16.9%. Several variables were investigated to determine potential contributors to the observed overestimation. Stratification of the data based on the mean sirolimus concentrations determined by both assays yielded no statistically significant differences in bias between concentration subgroups within the clinically relevant range. Multiple linear regression analysis identified HPLC-MS sirolimus concentration ( P = 0.03), hemoglobin concentration ( P < 0.001), and time after transplantation ( P< 0.001) as significant variables in the prediction of overestimation by MEIA. Analysis of the effect of time after transplantation on overestimation yielded a statistically significant difference up to 6 months after transplantation (35.6% to 46.4%) compared with 9 (23.9%) and 12 months (24.4%). A relationship between hemoglobin concentration and time after transplantation may explain the reduction in bias observed after 6 months. Conclusion The MEIA overestimates sirolimus concentrations in renal transplant patients compared with HPLC-MS. The clinical importance of this observed overestimation requires further investigation.