Diagnostic and therapeutic decisions in medical practice are still generally based on blood concentrations of drugs and/or biomolecules despite the knowledge that biochemical events and pharmacological effects usually take place in tissue rather than in the bloodstream. Microdialysis is a semi-invasive technique that is able to measure concentrations of the free, active drug or endogenous compounds in almost all human tissues and organs. It is currently being used to monitor brain metabolic processes and quantify tissue biomarkers, and determine transdermal drug distribution and tissue pharmacokinetics, confirming its importance as a widely used sampling technique in clinical drug monitoring and drug development as well as therapy and disease follow-up, contributing to rationalizing drug dosing regimens and influencing the clinical decision-making process.