Cell migration is a process that underlies the development and maintenance of multicellular organisms, with profound implications in various pathologies. The study of cell migration is fundamental in various fields of basic biology and pharmaceutical development. Wound healing assay is an indirect way to assess cell migration. Conventional methods, such as the scratch test, are inexpensive and easy to execute but have the disadvantages of being poorly reproducible and difficult to perform on a high-throughput scale. Meanwhile, commercial strategies are expensive. In the present work, we developed a lab-made wound healing assay device that is inexpensive, easy to handle, and reproducible. We designed 3D-printed stoppers compatible with cell culture in 96-well plates. These stoppers did not affect HaCaT cells viability. The stopper-produced initial wound size was reproducible on a high-throughput scale. Also, stoppers demonstrated their effectiveness to evaluate cell migration and allowed differentiating treatments with and without fetal bovine serum. Finally, proliferation assay was determined in this wound healing model. In conclusion, our lab-made 3D-printed stopper-based assay is a more economical alternative to currently available strategies for developing reproducible, high-throughput assays to assess cell migration and proliferation.