Sirolimus is used on patients after solid organ transplantation and on lymphangioleiomyomatosis (LAM) patients, and therapeutic drug monitoring is required in clinical practice. We have previously reported an accurate method for quantitative determination of sirolimus, but its sample preparation step was complicated. In this study, we developed a modified liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method for sirolimus quantification. A supported liquid extraction cartridge was used to purify sirolimus from whole blood and ion suppression was mostly prevented. The validation results fulfilled the acceptable criteria. This method was compared with the antigen conjugated magnetic immunoassay (ACMIA) and our previously reported method, using whole blood samples from LAM patients. Comparison of the Bland-Altman plots of the currently developed method and the previous method revealed no significant difference between the two methods (mean bias, -2.02%; 95% CI, -7.81 to 3.78). The values obtained by using ACMIA were significantly higher than those obtained by using the current method by 13.87% (95% CI, 6.49 to 21.25) due to cross-reactivity. The degree of cross reactivities in LAM patients and in organ transplant patients were similar, and our LC/ESI-MS/MS method precisely measured blood concentrations of sirolimus.