Extracellular vesicles (EVs) are cell-to-cell communication tools. Migrasomes are recently discovered microscale EVs formed at the rear ends of migrating cells, and thus are suggested to be involved in communicating with neighboring cells. In cell culture, peptide scaffolds on substrates have been used to demonstrate cellular function for regenerative medicine. In this study, we evaluated peptide scaffolds, including cell penetrating, virus fusion, and integrin-binding peptides, for their potential to enable the formation of migrasome-like vesicles. Through structural and functional analyses, we confirmed that the EVs formed on these peptide-modified substrates were migrasomes. We further noted that the peptide interface comprising cell-penetrating peptides (pVEC and R9) and virus fusion peptide (SIV) have superior properties for enabling cell migration and migrasome formation than fibronectin protein, integrin-binding peptide (RGD), or bare substrate. This is the first report of migrasome formation on peptide-modified substrates. Additionally, the combination of 95% RGD and 5% pVEC peptides provided a functional interface for effective migrasome formation and desorption of cells from the substrate via a simple ethylenediaminetetraacetic acid treatment. These results provide a functional substrate for the enhancement of migrasome formation and functional analysis.