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BACKGROUND Monitoring immunosuppressant levels, such as mycophenolic acid (MPA), cyclosporin A (CsA), and tacrolimus (TAC), in peripheral blood mononuclear cells (PBMCs) could be useful in organ transplant patients administered individualized therapy. The authors developed an LC-MS/MS assay technique to simultaneously determine immunosuppressant levels in PBMCs and assess their pharmacokinetics (PK) in Chinese renal allograft recipients. METHODS PBMCs were isolated from the whole blood of 27 Chinese renal transplant patients using Ficoll-Paque Plus solution, cell nuber was determined; acetonitrile treatment for protein precipitation, and gradient elution was performed on an Agilent Eclipse XDB-C18 column (3.5 μm, 2.1 mm×100 mm) with mobile phase: water and methanol (containing 2 mM ammonium formate); flow rate: 0.3 mL·min. RESULTS The calibration curves of MPA, CsA, and TAC had a linear range (ng mL): 0.098-39.2 (r=0.9987), 0.255-102 (r=0.9969), and 0.028-11.2 (r=0.9993), respectively. The extraction effects, matrix effects, and mean relative recovery of these immunosuppresants were 70.4-93.2%, 72.7-96.5%, and 90.1-112.4%, respectively. The within-day and between-day coefficients of variation were <15%. The AUC0-12 of MPA in PBMCs correlated well with those in plasma. The level of MPA, CsA and TAC in PBMCs might be more stable during dosing interval. CONCLUSION The derived LC-MS/MS assay is suitable for simultaneously monitoring different immunosuppressants in PBMCs. PK of MPA, CsA, and TAC displayed considerable inter-individual variability. Intracellular monitoring of immunosuppressants may facilitate individualized therapy for renal allograft recipients.