Tacrolimus, sirolimus, everolimus and Cyclosporine A are immunosuppressive drugs used in organ transplantation in order to prevent the rejection of transplanted organs and tissues. In addition, they are used in the therapy of autoimmune diseases. Due to high pharmacokinetic variability, narrow therapeutic indexes and many potential drug-drug interactions, close monitoring of blood concentrations is required to prevent rejection and minimize toxicity. The quantitative determination of these immunosuppressants should be performed only with LC-MS/MS, because the immunological tests vary considerably in terms of selectivity and specificity, and sometimes also detect inactive metabolites.