Creating multicellular tumor spheroids is critical for characterizing anticancer treatments since it may provide a better model than monolayer culture of in vivo tumors. Moreover, continuous dynamic perfusion allows the establishment of physiologically relevant drug profiles to exposed spheroids. Here we present a physiologically inspired design allowing microfluidic self-assembly of spheroids, formation of uniform spheroid arrays, and characterizations of spheroid dynamics all in one platform. Our microfluidic device is based on hydrodynamic trapping of cancer cells in controlled geometries and the formation of spheroids is enhanced by maintaining compact groups of the trapped cells due to continuous perfusion. It was found that spheroid formation speed (average of 7 h) and size uniformity increased with increased flow rate (up to 10 μl min⁻¹). A large amount of tumor spheroids (7,500 spheroids per square centimeter) with a narrow size distribution (10 ± 1 cells per spheroid) can be formed in the device to provide a good platform for anticancer drug assays.