Supramolecular hydrogels assembled from amino acids and peptide‐derived hydrogelators have shown great potential as biomimetic three‐dimensional (3D) extracellular matrices because of their merits over conventional polymeric hydrogels, such as non‐covalent or physical interactions, controllable self‐assembly, and biocompatibility. These merits enable hydrogels to be made not only by using external stimuli, but also under physiological conditions by rationally designing gelator structures, as well as in situ encapsulation of cells into hydrogels for 3D culture. This review will assess current progress in the preparation of amino acids and peptide‐based hydrogels under various kinds of external stimuli, and in situ encapsulation of cells into the hydrogels, with a focus on understanding the associations between their structures, properties, and functions during cell culture, and the remaining challenges in this field. The amino acids and peptide‐based hydrogelators with rationally designed structures have promising applications in the fields of regenerative medicine, tissue engineering, and pre‐clinical evaluation. Interest in amino acids and peptide‐based supramolecular hydrogels for 3D cell culture is increasing. This review assesses current progress in the preparation of these hydrogels under external stimuli and in situ cell encapsulation into the hydrogels, with a focus on understanding the associations between their structures, properties, and functions during cell culture and the remaining challenges.