A nonextraction homogeneous immunoassay (CEDIA Cyclosporine Plus Assay) has been developed for the measurement of cyclosporine in predose (trough) and post-dose (C2 to C8) whole-blood samples. The method includes a low-range assay that measures cyclosporine from 25 to 450 ng/mL in pre-dose samples and a high-range assay that detects cyclosporine from 450 to 2000 ng/mL in post-dose samples. The high-range assay allows a direct measurement of post-dose samples without a dilution step. Alternatively, post-dose samples can be correctly measured by the low-range assay following a twofold dilution. Using an NCCLS precision protocol, the assay exhibited less than 10% CV or error less than the functional sensitivity. Functional sensitivity of the low-range assay was demonstrated at 20 ng/mL cyclosporine. Cross-reactivity was measured in the presence of cyclosporine and was found to be 4.4%, 19.8%, 16.4%, 0.9%, 1.0%, and 1.6% for metabolites AM1, AM9, AM4n, AM19, AM4n9, and AM1c, respectively. When 53 samples were evaluated using an HPLC method, the three most significant cross-reactive metabolites, AM1, AM4n, and AM9, exhibited an average concentration profile of 123%, 19%, and 0.06% of the parent cyclosporine, respectively. The average total contribution to cyclosporine quantification from these metabolites was estimated at 7.2% based on the percentage cross-reactivity of each metabolite in the CEDIA assay and the concentration of each metabolite as determined by HPLC. The method comparison study revealed a linear regression correlation of CEDIA = 1.095 x HPLC + 6.6, r = 0.972, for the low-range assay, and CEDIA = 1.018 x HPLC - 36.4, r = 0.968, for the high-range assay. In conclusion, the CEDIA Cyclosporine Plus Assay is a precise and accurate method for quantification of cyclosporine in pre-dose and post-dose samples.