<p>The recent development of the kidney organoids provides a biochemical platform for investigating disease mechanism and drug screening. These three-dimensional constructs summarize key aspects of the human kidney, aiding in the study of complex disease processes. However, the conventional in vitro models fail to fully reflect the complexity of native tissues. To address this issue, there is a need to develop the novel culture systems that support physiologically improved kidney organoid differentiation. In this study, we investigate the efficacy of a microfluidic gradient chip system in enhancing the differentiation, maturation, and functionality of normal and Fabry disease kidney organoids. The results demonstrate that the fluidic flow promotes superior vascularization, glucose uptake, and gene expression associated with kidney maturation as compared to static culture conditions. Moreover, the increase in expression of specific markers and upregulation of kidney-related genes in microfluidic chip systems effectively enhance the differentiation, maturation, and functional potential of both normal and Fabry disease kidney organoids. Furthermore, the microfluidic gradient chip systems demonstrate improved sensitivity in detecting therapeutic effects, such as the restoration of the physiological characteristics after treatment of enzyme replacement therapy and oxidative stress. Furthermore, the microfluidic gradient chip developed in this study facilitates the physiological improvement of normal and Fabry disease kidney organoid differentiation and maturation. Therefore, this physiologically enhanced kidney organoid model could be used as a novel in vitro culture system for drug screening applications.</p>