The vasculature of the brain is made up of specialized endothelial cells that form a tight blood-brain barrier (BBB). Weakening of the junctional proteins can lead to BBB disruption. This can have detrimental effects on patients’ health, as the homeostatic environment of the brain becomes compromised.The MIMETAS OrganoPlate® is a microfluidic platform that enables perfused, 3D culture of up to 96 tissues in parallel in a membrane-free manner. The microfluidic channels allow growth of perfused tubular endothelial structures. Furthermore, the system allows for automation, enabling higher throughput than other microfluidic systems.To create a BBB model in the OrganoPlate, primary human brain microvascular endothelial cells (HBMECs) are seeded in the microfluidic channel against a collagen-I extracellular matrix. Under perfusion, the brain endothelial cells form confluent tubular structures, accessible from both the apical and basolateral sides. The BBB-on-a-chip model was characterized by immunostaining and shows expression of adherens junction proteins, as well as tight junction proteins. Expression of relevant BBB influx and efflux transporters were confirmed via PCR.Using fluorescent assays, we demonstrate low permeability of the BBB on-a-chip model to small molecule sodium fluorescein (0.45 nm radius). Upon exposure to known toxic compounds, the BBB on-a-chip model shows compound-induced leakage in a concentration-dependant manner. The same trend was observed using time-lapse measurements of the BBB on-a-chip’s transendothelial electrical resistance (TEER) with an automated multichannel impedance spectrometer called the OrganoTEER®.This OrganoPlate BBB model serves as a versatile and easy-to-use platform for high-throughput applications including toxicity and transport studies.