This paper reports HPLC methodology for the first successful enantiomeric separations of 3-phenylacetylamino-2,6-piperidinedione (PAP), a naturally occurring peptide derivative used for inhibiting the growth of cancer tissues. The chiral separation of four related hydrolysates is also described. A commercially available tris-4-methylbenzoate cellulose (Chiralcel OJ) column was used as the chiral stationary phase, operated in the normal-phase mode. The results demonstrated that hydrolyzed products of PAP, each of which has a carboxylic acid functionality present in its structure, eluted in a reasonable time and are enantiomerically resolved only when a trace amount of organic acid is present in the mobile phase. Different alcohols (ethanol and isopropanol) and acid additives (trifluoroacetic acid, trichloroacetic acid and acetic acid) were evaluated. In general, for the separation of the acidic enantiomers, ethanol is superior to isopropanol and stronger acids enhance the resolution more effectively. However, chiral separation of PAP could only be accomplished with isopropanol in the mobile phase and no acidic additive was needed.