Conventional liver models include a 2D planar culture of hepatocytes which is associated with a loss of metabolic activity and cells dedifferentiation. To overcome this limitation, the new tendencies is to use microfluidic devices to recapitulate the liver microenvironment. A relevant model should be suitable for 3D cultures due to the advantages it confers in the promotion of cell-cell and cell-matrix interactions. In addition, the model should be adapted for the co-culture of different cell types which has been proven to enhance hepatocytes functions. We previously developed a hepatocyte-on-chip model integrating a hyaluronic-based hydroscaffold with ECM components allowing the 3D organization of the cells in spheroid form. We successfully maintained the cells in culture under flow for 21 days. In this work, we are coupling the hepatocyte-on-chip device with liver sinusoidal endothelial cells (LSEC) in order to recreate the two major phenomena happening when a drug arrives to the liver: its passage through the liver sinusoids and the biotransformation reactions that occurs by the hepatocytes. First of all, the SK-Hep1 cell line is cultivated on a permeable membrane until confluence forming a monolayer of LSEC. At the same time, the HepG2/C3A cells, in contact with the hydroscaffold, assemble and form spheroids while attaching to the adhesion sites of the hydroscaffold. The cells in the two compartments are characterized separately for the membrane permeability and the spheroids formation. Then, the two systems were assembled on the “integrated insert in a dynamic microfluidic platform” to be linked to a peristaltic pump. The pump is used to circulate medium in closed loop through the LSEC barrier and then through the hepatocyte-seeded chamber. Then, cell viability, functionalities and metabolic activity for APAP are assessed. The results have shown that the monolayer of SK-Hep1 forms a barrier for the passage of drugs which may modulate the metabolic activity of the HepG2/C3a in the biochip. These results highlight the potential of our liver-on-chip model integrating co-cultured hepatocytes in 3D conformation with LSEC barrier in the enhancement of cells functions and the toxicity prediction and modulation.