The blood–brain barrier (BBB) with tightest junction separates the systemic circulation and brain micro-environment to protect the brain from insults, such as infections. The integrity of BBB is preserved by multi-structural and functional components. Increasing evidence indicates that BBB is used as an important marker measured in variety of pathological condition with large permeability leaks, such as brain tumors and multiple sclerosis, to more subtle disruption such as vascular diseases, cognitive decline, and dementia. Several imaging modalities are available to study disruption of the BBB. In this chapter, we described the protocols for nuclear imaging studies such as positron emission tomography (PET) and single photon emission computed tomography (SPECT) investigating BBB permeability in preclinical models.