Abstract Background Cyclosporine A and Tacrolimus are immunosuppressive drugs that are commonly used in solid organ transplant and are characterized by a relatively narrow therapeutic index, potential drug interactions and significant intra- and inter-individual variability. Therapeutic drug monitoring is therefore beneficial for optimizing dosage regimens, preventing rejection of the graft and reducing toxicities in transplant recipients receiving immunosuppressive drug therapy. A commercial assay (RECIPE ClinMass LC-MS/MS Advanced kit) for simultaneous measurement of immunosuppressants in whole blood using LC-MS/MS methodology has been adapted for assay performance evaluation in our laboratory on the Agilent 1290 Infinity II liquid chromatograph coupled to an Agilent 6470 triple quadrupole mass spectrometer. Our study evaluated the analytical performance of Cyclosporine A and Tacrolimus on the RECIPE assay and compared it against our existing in-house LC-MS/MS assay. Methods The RECIPE ClinMass® LC-MS/MS Advanced Immunosuppressants assay kit consist of mobile phase solvents, precipitation reagents, stable isotopic labeled internal standards, solid phase extraction and analytical columns, lyohilized whole blood calibrators and quality controls. Drug analytes were extracted from whole blood using single-step protein precipitation followed by on-line solid phase extraction on the LC, eluted on isocratic chromatography and detected on positive ESI MS ionisation mode. The RECIPE assay run time is under 3 min. Performance validation parameters included within/total assay imprecision, linearity, recovery, limits of detection (LOD)/quantification (LOQ) and carry-over. Method correlation studies using fresh patient whole blood specimens (n = 212 Cyclosporine A; n = 120 Tacrolimus) were performed on existing in-house assay on Agilent 6460 LC-MS/MS and the RECIPE assay on Agilent 6470 LC-MS/MS systems. Results Within run and total imprecision for Cyclosporine A and Tacrolimus on the RECIPE assay were determined to be ≤4.4% and ≤7.5% on the Agilent 6470 LC-MS/MS. The assay demonstrated linearity across the analytical measurement range up to 1500 ug/L for Cyclosporine A and up to 55 ug/L for Tacrolimus. Analytical recoveries obtained for both drugs ranged between 93%–100%. The LODs were assessed to be 13.0 ug/L and 0.70 ug/L for Cyclosporine A and Tacrolimus respectively. LOQs were assessed to be 22.7 ug/L and 1.30 ug/L for Cyclosporine A and Tacrolimus respectively. Results of carry-over studies were insignificant. Participation in the CAP Immunosuppressive Drugs External Quality Assessment Survey showed good agreement with LC-MS/MS peers. Method correlation with in-house assay demonstrated good agreement: (1) Cyclosporine A: Passing Bablok regression RECIPE = 0.94 (In-house) + 17.2, Bland Altman mean bias of +2.0%, Spearman correlation coefficient of 0.985; (2) Tacrolimus: Passing Bablok regression of RECIPE = 1.03 (In-house) - 0.2, Bland Altman mean bias of -0.2%, Spearman correlation coefficient of 0.988 Conclusion The RECIPE ClinMass® LC-MS/MS Immunosuppressants assay performed within manufacturer’s specifications except for LOD/LOQs which were determined at higher concentrations. The assay is adaptable to contemporary LC-MS/MS systems and confer benefits from rapid turnaround time (<3 min), simplified specimen preparation procedures and ready prepared reagents. The advantages allow enhancements in workflow efficiency and ease of staff training in our laboratory and the assay is suitable for routine clinical use in high workload clinical laboratories.