Most drugs exert their effects not within the plasma compartment, but in defined target tissues into which drugs have to distribute from the central compartment. Unfortunately, a complete and lasting equilibration between blood and tissue cannot always be taken for granted. Drug distribution processes may be characterized by a high intertissue- and intersubject variability and target site drug levels may substantially differ from corresponding plasma levels. Suboptimal target site concentrations may have important clinical implications, as it is a potential explanation for therapeutic failures. Therefore, determination of drug tissue penetration plays an important role in clinical drug development. In recent years, the assessment of tissue concentrations after administration of very low, sub-pharmacological drug amounts (microdosing) has attracted major interest in early clinical drug development, which calls for the availability of highly sensitive analytical methods. The present article will review the most important techniques that are currently available for studying drug disposition in humans. These can be classified as semi-invasive (microdialysis, MD) and non-invasive (positron emission tomography, PET, and magnetic resonance spectroscopy, MRS). We will discuss individual strengths and shortcomings of each method and provide some recent examples with particular focus on antiinfective and anticancer drugs. Whereas MD and MRS also lend themselves to the assessment of tissue-specific drug metabolism, PET usually does not provide metabolic information. For some drugs, such as the anticancer agents 5-fluorouracil and capecitabine, measurement of drug metabolites is particularly important as these represent the therapeutically active species.