The blood-brain barrier (BBB), a highly selective semipermeable membrane, often involves the protection of the brain from the blood milieu, aids selective transport, and also affects the metabolism of certain substances. Evaluating the BBB can give a better understanding of how it functions and can be manipulated to improve drug delivery to the brain. The BBB stands as a major roadblock in developing therapies for CNS diseases including neurodegenerative diseases and cerebrovascular diseases. Quantification of BBB permeability either by in vitro or in vivo methods is crucial in understanding brain disorders and developing strategies for drug delivery to CNS. Among several imaging modalities available to study the disruption of BBB, in this chapter, we described the latest protocols for nuclear imaging studies, namely, positron emission tomography (PET) and single photon emission computed tomography (SPECT) in preclinical models.