SummarySeveral dipeptides have been separated into stereoisomers (enantiomers, diastereoisomers, sequential isomers) by GC on the chiral stationary phase Chirasil-Val. Sufficiently volatile N-TFA-dipeptide methyl esters have been formed by derivatization at ambient temperature, thus avoiding racemization (or epimerization, respectively), and peptide cleavage. This novel approach has proven particularly useful for the determination of stereoisomeric composition of dipeptides in biological fluids.