Rapamycin (RAPA) is a carbocyclic lactone-lactam macrolide antibiotic with immunosuppressive properties (1, 2). In vitro studies have shown RAPA to be up to 100 times more potent than Cyclosporine A (2–5). Studies in a variety of animal transplant models have shown RAPA to prolong allograft survival (2, 6–12). A synergistic effect between RAPA and cyclosporine has been reported in vitro with human lymphocytes (13) and in vivo with rats (13), mice (14), and dogs (15). Federal regulatory agencies such as the Food and Drug Administration in the United States and the Health Protection Branch in Canada are placing increasing emphasis on therapeutic monitoring of drugs with narrow therapeutic windows (16). It is recommended that early establishment of standardized protocols that encompass analytical, pharmacodynamic, pharmacokinetic, and toxicokinetic aspects of therapeutic drug monitoring will facilitate the approval process of the drug, as well as provide better patient monitoring once the drug is released. However, in the majority of early preclinical studies with RAPA, the drug was administered on a dose-per-weight basis, with little regard given to determining the effective blood concentrations (2, 6–9). This was in part, due to a lack of both a suitable analytical method and information regarding the most optimal medium for monitoring the drug.