Aims Immunosuppressant and kidney function monitoring are crucial for kidney transplant recipient follow-up. Microsamples enable remote sampling and minimise patient burden as compared to... Show moreAims Immunosuppressant and kidney function monitoring are crucial for kidney transplant recipient follow-up. Microsamples enable remote sampling and minimise patient burden as compared to conventional venous sampling at the clinic. We developed a liquid chromatography-tandem mass spectrometry assay to quantify tacrolimus, mycophenolic acid (MPA), creatinine and iohexol in dried blood spot (DBS), and volumetric absorptive microsample (VAMS) samples. Methods The assay was successfully validated analytically for all analytes. Clinical validation was conducted by direct comparison of paired DBS, VAMS and venous reference samples from 25 kidney transplant recipients. Patients received iohexol 5-15 minutes before immunosuppressant intake and were sampled 0, 1, 2 and 3 hours thereafter, enabling tacrolimus and MPA area under the concentration-time curve (AUC) and creatinine-based and iohexol-based glomerular filtration rate (GFR) estimation. Method agreement was evaluated using Passing-Bablok regression, Bland-Altman analysis and the percentages of values within 15-30% of the reference (P-15-P-30) with a P-20 acceptance threshold of 80%. Results For DBS samples, method agreement was excellent for tacrolimus trough concentrations (n = 25, P-15 = 92.0%) and AUCs (n = 25; P-20 = 95.8%) and adequate for creatinine-based GFR trend monitoring (n = 25; P-20 = 80%). DBS-based MPA AUC assessment showed suboptimal agreement (n = 16; P-20 = 68.8%), but was considered acceptable given its P-30 of 100%. The assay performed inadequately for DBS-based iohexol GFR determination (n = 24; P-20 = 75%). The VAMS technique generally showed inferior performance, but can be considered for certain situations. Conclusion The assay was successfully validated for tacrolimus, MPA and creatinine quantification in DBS samples, enabling simultaneous remote kidney function trend monitoring and immunosuppressant therapeutic drug monitoring in kidney transplant recipients. Show less