Tissue-engineered in vitro models have the potential to be used for investigating inflammation in the complex microenvironment found in vivo. We have developed an in vitro model of hepatic tissue that facilitates real-time monitoring of endothelium activation in liver tissue. This was achieved by creating a layered coculture model in which hepatocytes were embedded in collagen gel and a reporter clone of endothelial cells, which synthesizes green fluorescent protein in response to nuclear factor-kappa B (NF-κB) activation, was overlaid on top of the gel. The efficacy of our approach was established by monitoring in real time the dynamics of NF-κB-regulated fluorescence in response to tumor necrosis factor α. Our studies revealed that endothelial cells in coculture with hepatocytes exhibited a similar NF-κB-mediated fluorescence to both pulse and step stimulation of lipopolysaccharide. By contrast, endothelial cells in monoculture displayed enhanced NF-κB-regulated fluorescence to step in comparison to pulse lipopolysaccharide stimulation. The NF-κB-mediated fluorescence correlated with endothelial cell expression of NF-κB-regulated genes such as intercellular adhesion molecule 1, vascular cell adhesion molecule 1, and E-Selectin, as well as with leukocyte adhesion. These findings suggest that our model provides a powerful platform for investigating hepatic endothelium activation in real time.