The use of combinations of immunosuppressant drugs is considered the therapeutic gold standard for post-allogeneic hematopoietic stem cell transplantation (HSCT) to prevent serious complications such as graft versus host disease and rejection. However, these drugs have a narrow therapeutic index and wide inter-individual pharmacokinetic fluctuations, resulting in unpredictable levels of drugs in the blood. Systemic concentrations of several immunosuppressive drugs have been correlated with their efficacy and potential life-threatening complications, thus supporting the need for a pharmacokinetic monitoring. Some chromatographic techniques have been proposed for the simultaneous monitoring of multiple immunosuppressive drugs, but none offers the possibility of measuring cyclosporine, tacrolimus, methotrexate, prednisone, prednisolone, and methylprednisone in addition to total and free mycophenolic acid (MPA) and its phenolic (MPAG) and acyl (AcMPAG) glucuronides. To address this critical deficiency, a liquid chromatography–coupled tandem mass spectrometry method was developed for the quantification of circulating levels of these multiple immunosuppressant drugs. Linearity, precision and accuracy were validated within the typical therapeutic range of concentrations for each compound. The method could measure individual drugs with high sensitivity, accuracy (bias <14%), and reproducibility (CV <12.8%). Its clinical application was validated by measuring levels of these drugs in samples obtained from HSCT recipients treated with combined immunosuppressive drug therapy. Our results indicate that this approach is suitable for simultaneous determination of in vivo levels of immunosuppressive drugs commonly used in combined therapies. This method requires a small-volume peripheral blood sample, an attribute that may be beneficial to HSCT patients in the clinic. Indeed, owing to the frequent administration of myeloablative conditioning regimens in the pre-transplantation period, the recovery of hematologic function is slow and gradual after engraftment. It is therefore favorable to limit blood sample collection in the immediate post-transplantation period. This method may be convenient for large-scale clinical studies aimed at improving and optimizing patient outcomes. It could also be useful in therapeutic drug monitoring to promote the efficient use of immunosuppressive drugs and thus to limit life-threatening complications related to non-optimal exposure to immunosuppressive medications.