The blood-brain barrier (BBB) and drug delivery: In our mod societies, the prevalence of nervous system disorders is increasing in relation to the aging population. To overcome this problem, sev-eral interesting pharmacons and biomolecules are introduced and tested each day. The biggest obstacle to delivering drugs into the brain parenchyma is the presence of the BBB (Toth et al., 2011). The BBB is a regulatory interface, which limits free transport of large and small molecules between the systemic circulation and the central nervous system. This regulation enables proper neuronal function and protection from outside toxic insults as well as main-tenance of a stable ionic homeostasis. The morphological basis of the BBB is the monolayer of brain endothelial cells (BECs) in the cerebral microvessels. The adult human brain contains more than 600 km of capillaries, which differ fundamentally from other endo-thelial interfaces in the body. The tight inter-endothelial junctions connecting the cells reduce paracellular permeation of ions and other small hydrophilic solutes as well as larger molecules. Conse-quently, molecules have to enter the brain via transcellular trans-port mechanisms, but this access is also subjected to restrictive control. The metabolic and enzymatic barrier, formed by a unique expression patt of enzymes and efflux pumps at the luminal membrane of BEC, limits BBB penetration of lipophilic drugs and other xenobiotics. Thus, the supply of essential nutrients to brain cells is tightly controlled via the vesicular-mediated transcellular transport mechanism. Transcytosis of larger molecules such as peptides and proteins are initially endocytosed by absorptive- and receptor-mediated mechanisms and then transcytosed via sub-cellular vesicles. This regulated vesicular transport is also known as absorptive- and receptor-mediated transcytosis (Abbott et al., 2010). In addition to essential supplements, cell surface receptors are considered a potential gate for targeted delivery of large drugs to the brain. In the last decade, several publications have focused on the transferrin receptor as a target for bispecific antibodies and nanoparticles with pharmaceutical effect (Freskg?rd and Urich, 2017). Additionally, the cross-talk among endothelial cells and neighboring cells such as astroglia, pericytes, microglia, and neu-rons should be mentioned, which induce a unique barrier pheno-type in BEC. This interaction is important for drug delivery, as it is known to affect expression of tight junction molecules, receptors, and transporters, as well as influence the subcellular vesicular sys-tem (Abbott et al., 2010). In this paper, we introduce high content screening microscopy as an approach to analyze the subcellular ve-sicular structure and the trafficking system of the BBB in vitro. The method is particular useful to describe and compare differences between different culture set-ups.