A microfluidic device with 3-D hydrogel cell cultures has been developed to test the cytotoxicity of anti-cancer drugs while reproducing multi-organ interactions. In this device, a micro cell culture analog (µCCA), cells embedded in 3-D hydrogels are cultured in separate chambers representing the liver, tumor, and marrow, which are connected by channels mimicking blood flow. While the microfluidic network provides a platform for mimicking the pharmacokinetic and pharmacodynamic profiles of a drug in humans, the 3-D hydrogel provides a more physiologically realistic environment to mimic the tissue than monolayer culture. Colon cancer cells (HCT-116) and hepatoma cells (HepG2/C3A) were encapsulated in Matrigel and cultured in the tumor and the liver chamber in a µCCA, respectively. Myeloblasts (Kasumi-1) were encapsulated in alginate in the marrow chamber; a stiffer hydrogel was necessary to prevent cell migration out of the matrix. The cytotoxic effect of Tegafur, an oral prodrug of 5-fluorouracil (5-FU), on each cell line was tested using the µCCA with cell-embedded hydrogel. The comparison of experimental results using a 96-well microtiter plate and a µCCA demonstrated that the µCCA was able to reproduce the metabolism of Tegafur to 5-FU in the liver and consequent death of cells by 5-FU, while the cultures in a 96-well microtiter plate were unable to do so. The µCCA utilizing 3-D hydrogel cell cultures has potential as a platform for pharmacokinetic-based drug screening in a more physiologically realistic environment.