A novel, reversed-phase high-performance liquid chromatographic (HPLC) method is described for the analysis of sirolimus (SRL) in whole blood. The samples were purified by precipitating blood matrix with zinc sulfate, SRL was then extracted with acetone followed by solid-phase extraction. The method was linear over a range of 1-100 ng/ml and the lower limit of quantification was 2.5 ng/ml. The coefficient of variation (within day) was below 8.0% for the lowest SRL concentration. The day-to-day coefficient of variation was below 6.6%. The assay did not show interference peaks with immunosuppressive drugs commonly given to transplant patients. With the simplified extraction procedure described, 60 samples (including controls and calibration curve) can be quantified in a day. The sensitivity and rapidity of this analytical procedure makes it useful for routine therapeutic monitoring of SRL.