Everolimus was developed for use with cyclosporine as a new immunosuppressant in the prevention of rejection in transplant patients. It binds to FK binding protein and modulates the activities of the mammalian Target of Rapamycin following entry into cytoplasm. A reliable and accurate measurement of everolimus blood concentrations is vital in kidney transplant patients. The purpose of this study was to test for the potential cross-reactivities of the major hydroxylated everolimus metabolites with the immunoassay developed to measure everolimus in the blood of transplant patients. Everolimus is primarily metabolized by the cytochrome P4503A enzymes of the liver and small intestine. The major everolimus metabolites in the blood of kidney transplant patients (n=133 samples) have been identified as 45/46-hydroxy, 24-hydroxy, and 25-hydroxy everolimus. To test the cross-reactivity of the three major metabolites, everolimus was incubated with human liver microsomes for 15 minutes in vitro, which resulted in the formation of 9 hydroxylated metabolites. The metabolites of interest, 45/46-hydroxy, 24-hydroxy, and 25-hydroxy everolimus, were identified by Mass Spectrometry (MS)-ion trap MS/MS analysis and the blood samples from kidney graft patients were extracted and run under the same chromatographic conditions. Comparison of the MS/MS spectra from metabolites from patient blood samples with the metabolites from human liver microsome incubations in vitro, confirmed that the structures of the major hydroxy everolimus metabolites in blood were identical to those produced in vitro. An HPLC/UV assay was used to isolate the hydroxy metabolites, and the metabolite purities were confirmed by HPLC/UV and LC/MS analysis, which indicated that the metabolites were ≥99% free of other everolimus derivatives, and most importantly, none of the samples contained detectable amounts of everolimus (lower limit of detection of LC/MS assay: 0.1 ng/mL). The three metabolites were separately spiked in blood and prepared for further for cross-reactivity testing with a clinical immunoassay. 3-hydroxy everolimus metabolites present in blood of kidney transplant patients were produced and isolated in vitro with sufficient purity and quantity for cross reactivity testing with clinical immunoassay. In conclusion, we have established a reliable and accurate assay for everolimus. Furthermore, this study indicates that everolimus has a narrow therapeutic range and requires close therapeutic drug monitoring for optimal immunosuppression in transplant patients.