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Therapeutic drug monitoring (TDM) of immunosuppressants is essential to optimize patient care after organ transplantation. In blood, most immunosuppressive drugs are bound to plasma proteins or located inside blood cells. However, it is generally assumed that only protein-unbound (free) drug concentrations are pharmacologically active and could therefore better reflect the clinical outcome. Study data are still limited due to lacking rapid analytical methods. Therefore, a simple multiplex method for direct measurement of free cyclosporine A (CsA) and mycophenolic acid (MPA) has been developed.The sample preparation included ultracentrifugation, followed by liquid–liquid extraction. Stable isotope labeled analogues of CsA and MPA were used as internal standards. The LC-MS/MS analysis was performed on a triple quadrupole mass spectrometer in the multiple reaction monitoring mode. The validated assay was used in a study of 40 blood samples from kidney transplant patients.The lower limits of quantification were 0.1 (CsA) and 0.5 ng/mL (MPA). Assay linearity was confirmed in the concentration ranges of 0.1–10.0 ng/mL (CsA) and 0.5–100 ng/mL (MPA). For both analytes, inaccuracy was ≤9.8% and imprecision was ≤7.8%. The extraction efficiency ranged between 91% and 96%. In the patient samples the average free CsA and MPA fractions were 5.8% (2.1%–16.8%) and 1.2% (0.5%–2.4%) respectively.A reliable and highly sensitive LC-MS/MS method as a new suitable tool for measuring protein-unbound CsA and MPA has been developed, validated and applied in kidney transplant patient samples. Now, larger studies can be conducted to investigate the benefit of free drug monitoring in transplant recipients.