Positron emission tomography (PET) imaging using microdoses of radiolabeled drug tracers is gaining increasing acceptance in modern clinical drug development. This approach is unique in that it allows for direct quantitative assessment of drug concentrations in the tissues targeted for treatment, thereby bridging the gap between pharmacokinetics and pharmacodynamics. Current applications of PET in anticancer, anti-infective and central nervous system drug research are reviewed herein. Situated at the interface of preclinical and clinical drug testing, PET microdosing is a powerful and highly innovative tool for pharmaceutical development.