The blood-brain barrier (BBB) is a complex and essential structure that protects the brain from potentially harmful substances in the bloodstream. It is a selective barrier that only allows certain molecules to pass through to the brain while blocking others. Research into the BBB is crucial for understanding neurological diseases and developing new treatments. Zebrafish have become a popular model organism for BBB research due to their transparent embryos and larvae, which allow for easy visualization and manipulation of the BBB. In this chapter, we will explore the use of zebrafish in BBB research, the advantages and limitations of this model organism, and the methods that have been made possible through the use of zebrafish in this field. The principal techniques for permeability assays include (i) the measure of different tracers across the BBB in zebrafish embryos or adults, (ii) fluorescent imaging of genes related to BBB endothelial cells integrity, (iii) electron microscopy able to understand ultrastructure and morphology of the BBB in zebrafish, and (iv) orthotopic xenograft model used to evaluate drugs to cross the BBB and target brain tumors. Advances in experimental techniques and strategies using zebrafish can contribute to the understanding of the BBB and its interface between the systemic environment and the central nervous system in different physiological and pathological conditions.