Blood vessel-on-a-chip models aim at reproducing vascular functions. However, very few efficient methods have been designed to address the need for biological replicates in medium- to high-throughput screenings. Here, vessels-on-chip were designed in polydimethylsiloxane-glass chips using the viscous finger patterning technique which was adapted to create channels with various internal diameters inside a collagen solution and to simultaneously seed cells. This method was refined to create blood vessels composed of two concentric, distinct, and closely appositioned layers of human endothelial and perivascular cells arranged around a hollow lumen. These approaches allowed the formation of structurally correct blood vessels-on-chips which were constituted of either only endothelial cells or of both cell types in order to distinguish the vascular barrier reactivity to drugs in the presence or not of perivascular cells. The established vessels showed a tight vascular barrier, as assessed by immunostaining of the adherens junctions, and were reactive to the natural vasopermeant thrombin and to inflammatory cytokines. The presence of perivascular cells markedly increased the tightness of the vascular barrier and lowered its response to thrombin. The design allowed us to simultaneously challenge in real-time several tens of 3D-reconstituted, multicellular blood vessels in a standard multiwell plate format suitable for high-throughput drug screening.