A rapid small-scale peptide synthesis method was developed based on in vitro translation system. It enables us to obtain peptides within a day. Comparing the biological activity of in vitro synthesized peptides with that of chemically synthesized ones, the activity of both peptides was confirmed to be equivalent. The recovered peptides were assumed correct in their sequence through MS analysis and estimated to be more than pmoles/run. Moreover, this method could produce highly hydrophobic peptide, otherwise unavailable.