One important application of tissue engineering is to provide novel in vitro models for cell-based assays. Perfusion microbioreactor array provides a useful tool for microscale tissue culture in parallel. However, high-throughput data generation has been a challenge. In this study, a 4 x 4 array of perfusion microbioreactors was developed for plate-reader compatible, time-series quantification of cell proliferation, and cytotoxicity assays. The device was built through multilayer soft lithography. Low-cost nonwoven polyethylene terephthalate fibrous matrices were integrated as modular tissue culture scaffolds. Human colon cancer HT-29 cells with stable expression of enhanced green fluorescent protein were cultured in the device with continuous perfusion and reached a cell density over 5 x 10(7) cells/mL. The microbioreactor array was used to test a chemotherapeutic drug 5-FU for its effect on HT-29 cells in continuous perfusion 3D culture. Compared with conventional 2D cytotoxicity assay, significant drug resistance was observed in the 3D perfusion culture. (C) 2010 American Institute of Chemical Engineers Biotechnol. Prog., 26: 1135-1144, 2010