Tacrolimus is amongst the most used immunosuppressants after transplantation. To avoid organ rejection or toxicity, Therapeutic Drug Monitoring (TDM) is recommended, which involves the measurement of tacrolimus concentrations in whole blood. Whole blood is the preferred matrix because of the large protein and erythrocyte binding. This project -coined TDM 2.0- wishes to move on where conventional TDM, using conventional liquid blood samples, stops. Microsampling -i.e. the collection of minute amounts of blood to generate dried blood samples, allowing home sampling- will be used throughout this project. As a first aim we wish to overcome the sensitivity challenge associated with TDM of tacrolimus, when starting from a microsample, by employing different innovative bioanalytical strategies. Despite the indisputable value of TDM, it is important to note that tacrolimus concentrations do not always adequately predict organ rejection. Therefore, complementary to traditional TDM, we will focus on CYP3A5 phenotyping, calcineurin activity measurement and biomarker determination. As a second aim phenotyping of CYP3A5 will be performed by combining microsampling with sparse sampling for quantitative determination of tacrolimus. This will involve two patient studies. As a third and fourth aim the potential of enzyme activity determination of the tacrolimus molecular target calcineurin will be evaluated, as will be the determination of protein biomarkers in dried blood microsamples.