Although microfluidic cell culture systems are versatile tools for cellular assays, their use has yet to set in motion an evolutionary shift away from conventional cell culture methods. This situation is mainly due to technical hurdles: the operational barriers to the end-users, the lack of compatible detection schemes capable of reading out the results of a microfluidic-based cellular assay, and the lack of fundamental data to bridge the gap between microfluidic and conventional cell culture models. To address these issues, we propose a high-throughput, perfusion, three-dimensional (3-D) microfluidic cell culture system encompassing 30 microbioreactors. This integrated system not only aims to provide a user-friendly cell culture tool for biologists to perform assays but also to enable them to obtain precise data. Its technical features include (i) integration of a heater chip based on transparent indium tin oxide glass, providing stable thermal conditions for cell culturing; (ii) a microscale 3-D culture sample loading scheme that is both efficient and precise; (iii) a non-mechanical pneumatically driven multiplex medium perfusion mechanism; and (iv) a microplate reader-compatible waste medium collector array for the subsequent high throughput bioassays. In this study, we found that the 3-D culture sample loading method provided uniform sample loading [coefficient of variation (CV): 3.2%]. In addition, the multiplex medium perfusion mechanism led to reasonably uniform (CV: 3.6-6.9%) medium pumping rates in the 30 microchannels. Moreover, we used the proposed system to perform a successful cell culture-based chemosensitivity assay. To determine the effects of cell culture models on the cellular proliferation, and the results of chemosensitivity assays, we compared our data with that obtained using three conventional cell culture models. We found that the nature of the cell culture format could lead to different evaluation outcomes. Consequently, when establishing a cell culture model for in vitro cell-based assays, it might be necessary to investigate the fundamental physiological variations of the cultured cells in different culture systems to avoid any misinterpretation of data. As a whole, we have developed an integrated microfluidic cell culture system that overcomes several technical hurdles commonly encountered in the practical application of microfluidic cell culture systems, and we have obtained fundamental information to reconcile differences found with data acquired using conventional methods.