The blood-brain barrier (BBB) is a highly specialized structure that functions as a critical interface, isolating the central nervous system (CNS) from the body’s systemic circulation. It is primarily composed of brain endothelial cells (BECs), which are characterized by a scarcity of pinocytic vesicles, lack of fenestrae, and the presence of paracellular tight junctions (TJs) and adherens junctions (AJs). The BBB works closely with other brain cells, forming a dynamic and complex cellular structure known as the neurovascular unit (NVU). The NVU comprises neurons, interneurons, astrocytic endfeet, microglia, oligodendrocytes, basal lamina covered with smooth muscular and pericytes, endothelial cells, extracellular matrix, and circulating blood components. The NVU facilitates intercellular communication, shaping the characteristics of the BBB and its response to physiological stimuli. This interaction enables the regulation of vascular permeability, cerebral blood flow, and immune responses based on neural activity, all essential for maintaining homeostasis in the CNS. This communication is achieved through transporters and also from molecules that are produced and secreted by cells of the brain interfaces. Finally, cells of the BBB respond to signals from the CNS or blood compartments, which may stimulate alterations in their barrier, transport, and secretory functions. A comprehensive understanding of the BBB’s role is crucial for mitigating its dysfunction in disease states while simultaneously identifying strategies to facilitate drug delivery across the barrier. This BBB book is focused on methods and protocols to evaluate BBB’s physiology, biochemistry, and molecular biology through innovative and relevant in vivo, ex vivo, and in vitro models for addressing these scientific challenges and advancing therapeutic interventions for diseases that affect the brain.