The human immune system represents a highly complex multicellular network that protects the organism against the environment and pathogens. Within this system, different immune cells communicate with each other, as well as with adjacent organs and tissues, using an impressive network of regulatory signals. This inherent complexity makes it rather difficult to mimic these processes in vitro. Unpredictable drug-induced side effects can be the consequence when moving from preclinical animal models into clinical phase. Therefore, there is a demand for more elaborate in vivo like human cell culture models. In this study, an in vitro co-culture model consisting of Caco-2 human gut epithelial cells and human whole blood representing the immune system is applied to investigate the intestinal absorption of anti-inflammatory drugs and the subsequent modulation of the immunoregulatory signaling processes. By using blood of different donors, the individuality of the immune system is integrated into the overall analysis. The anti-inflammatory drugs prednisolone and ibuprofen were applied on top of the Caco-2 epithelial cells and alterations in the extracellular communication via cytokines and chemokines were visualized using miniaturized multiplexed sandwich immunoassays. Optionally, pretreatment of the Caco-2 epithelial cells with pro-inflammatory mediators can be used to modulate the epithelial barrier function similar to the situation observed during inflammatory conditions of the gut. The presented translational test system, consisting of differentiated Caco-2 intestinal epithelial cells and whole blood substantially improves preclinical screening of immunologically active drugs with respect to an approximation of the human "in vivo" conditions.