In this paper, we examined the possibility of using conventional Edman degradation with phenyl isothiocyanate for the simultaneous determination of both the sequence and the d/l-configuration of amino acids in peptides. Boron trifluoride and HCl-methanol (1:10, v/v) were adopted as the cyclization/cleavage and conversion reagents instead of the respective use of anhydrous trifluoroacetic acid (TFA) and 20% aqueous TFA to suppress the amino acid residue racemization. The enantiomeric separation of 18 phenylthiohydantoin amino acids was achieved on two types of chiral stationary phases bonded with β-cyclodextrin. The proposed Edman procedure was applied to a synthetic β-amyloid 1-16 with all l-forms as a model peptide, affording the amino acid sequence and configuration determination up to 12 residues.