Microwell arrays are cell culture and imaging platforms to assess cells at a single cell level and in high-throughput. They allow the spatial confinement of single cells in microfabricated cavities on a substrate and thus the continuous long-term observation of single cells and their progeny. The recent development of microwell arrays from soft, biomimetic hydrogels further increases the physiological relevance of these platforms, as it substantially enhances stem cell survival and the efficiency of self-renewal or differentiation. This protocol describes the microfabrication of such hydrogel microwell arrays, as well as the cell handling and imaging.