4-Substituted prolines, especially 4-fluoroprolines, have been widely used in protein engineering and design. Here, we report a robust and stereoselective approach for the synthesis of (2S,4S)-methylproline starting from (2S)-pyroglutamic acid. Incorporation studies with both (2S,4R)- and (2S,4S)-methylproline into the Trx1P variant of the model protein thioredoxin of E. coli show that the stereochemistry of the 4-methyl group might be a key determinator for successful incorporation during ribosomal synthesis of this protein.