AbstractCancer cell spheroids have been shown to mimic in vivo tumour microenvironment and are therefore suitable for in vitro drug screening. Microfluidic technology can provide conveniences for spheroid assays such as high‐throughput, simplifying manual operation and saving reagent. Here, we propose a concentration gradient generator based on microfluidic technology for cell spheroid culture and assay. The chip consists of upper microchannels and lower microwells. After partitioning HepG2 suspension into the microwells with concave and non‐adhesive bottoms, spheroids can spontaneously form. By controlling the fluid replacement and flow in microchannels, the doxorubicin solution is diluted automatically into a series of concentration gradients, which spanning more than one order of magnitude. And then the effect of doxorubicin on spheroids is measured in situ by fluorescent staining. This chip provides a very promising approach to achieve the high‐throughput and standardized anti‐cancer drug screening in future.