As microfluidic technologies have been developed, many attempts have been made to co-culture with other kinds of cells in vitro with extracellular matrix (ECM) to mimic tumor microenvironment in vivo. While these approaches may provide more credible expectations for drug response compared with well-plate based assay, understanding the heterogeneous cellular response to drug treatment is limited by the average response of many cells in most conventional bulk-level assays. To solve these limitations, we developed single cell level screening platform to monitor heterogeneous anti-cancer drug response with interaction of tumor microenvironment. The device consists of a reservoir, a PDMS porous membrane and a glass substrate. With the two-layered PDMS membrane for single tumor entrapment and patterning of the stromal cells, the platform creates an environment that mimics tumor and stromal cells interaction in tumor microenvironment. We compared tumor apoptosis using JC-1 aggregates / monomers ratio in single cell level with anti-cancer chemotherapy in mono- and co-culture in the platform. From these experiments, we observed that heterogeneous pharmacodynamics (PD) of single tumor in mono-and co-culture against anti-cancer chemotherapy and co-culture of stromal cell made single tumor more resistant to chemotherapy. Furthermore, specific interesting single tumor and stromal cells can be retrieved for the further study of relevant drug resistance pathway. We anticipate this platform can be applicable to better understanding the signaling cascade of cellular networks for anti-cancer chemotherapy resistance.