Tumor spheroid formation in microwell arrays is a promising approach for high-throughput screening of chemotherapeutic agents. This method offers the advantage of better mimicking the complexities of tumors as compared to conventional monolayer culture systems. However, using these technologies to their full potential is hindered by the inability to seed the cells within the wells uniformly and with high yield and reproducibility. Moreover, standard manufacturing approaches for fabrication of microwell arrays rely on lithography and etching techniques, which are costly, labor-intensive, and time-consuming. Herein, we report on the development of self-filling microwell arrays (SFMAs) in which cells are directed from a loading chamber to microwells using inclined guiding channels. The SFMAs are fabricated by replica molding of three-dimensionally (3D) printed molds in agarose. We characterize the fabrication process, demonstrate the ability to culture breast adenocarcinoma MCF-7 and glioma U87 in SFMAs and perform drug toxicity studies. We envision that the proposed innovative approach opens avenues of opportunities for high-throughput three-dimensional cell culture for drug screening and disease modeling.