The 18 kDa translocator protein (TSPO) is an evolutionarily conserved transmembrane protein found embedded in the outer mitochondrial membrane. A secondary target for the benzodiazepine diazepam, TSPO has been a protein of interest for researchers for decades, particularly owing to its well-established links to inflammatory conditions in the central and peripheral nervous systems. It has become a key biomarker for assessing microglial activation using positron emission tomography (PET) imaging in patients with diseases ranging from atherosclerosis to Alzheimer's disease. This Account describes research published by our group over the past 15 years surrounding the development of TSPO ligands and their use in probing the function of this high-value target.