Simultaneous separation of 37 proteinogenic D/L-amino acids excluding proline with HPLC is generally difficult. In this study, two sets of o-phthalaldehyde (OPA)-based fluorescent diastereomers of D/L-amino acids were formed using two chiral thiols of N-acetyl-L-cysteine (NAC) and N-isobutyryl-L-cysteine (NIBC) independently. It is expected that obtained two sets of OPA-derivatized D/L diastereomers show different retention selectivity in reversed phase chromatography because NAC and NIBC have different hydrophobicities. The two independent HPLC methods were designed to obtain complementary separations for the two sets of fluorescent diastereomers of the proteinogenic D/L-amino acids. The obtained two complementary chromatograms provided complete determination for diastereomers of proteinogenic D/L-amino acids. After the optimization of HPLC separation conditions, two types of automated analytical procedures were investigated. One was for OPA/NAC and OPA/NIBC derivatization of amino acids using originally equipped pretreatment functions of auto-sampler. The other was for switching the two HPLC separation methods during the consecutive batch run. Through these re-consideration for automated procedures, finally obtained method afforded satisfactory peak area repeatability of 1.5%RSD or less and calibration curve linearity of 0.999 or greater for each diastereomers. The established methods were applied to the real liquor samples and successful results were obtained.