Therapeutic drug monitoring (TDM) of immunosuppressants is critical to achieving optimal patient care after solid organ transplantation. During the past years, high performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) has become an increasingly important tool for TDM of immunosuppressants due to its high specificity and sensitivity compared to other available technologies including immunoassays. This review is focused on the most recent discovery and development of HPLC-MS/MS in immunosuppressant TDM. The immunosuppressants covered in this review are cyclosporin A, tacrolimus, mycophenolic acid, sirolimus, and everolimus. The advantages and practical issues are discussed in detail. In conclusion, HPLC-MS/MS is considered superior to immunoassays for immunosuppressant TDM. However, careful selection and evaluation of internal standards and the solvents used for solution preparation and column elution are important for reliable assay performance.