A method of manufacturing a non-natural D- or L-amino acid derivative possessing a protected or non-protected thiol group and substituent R 1 at the β-position, wherein R 1 refers to the substituent moiety bound to the β carbon atom among side chain substituents that configure amino acids except when it is a hydrogen atom, comprising the following steps of: (I) carrying out the following reactions on an amino acid derivative to manufacture an amino acid derivative possessing a protected or non-protected thiol group at the β-position comprising D- and L-forms: (P) manufacturing an amino acid derivative possessing substituent R 1 and a leaving group L on the β carbon atom, wherein said step (P) comprises the following step (P-2): (P-2) reacting glycine with an aldehyde compound represented by R 1 CHO, (A) a reaction of introducing a protected or non-protected thiol group at the β carbon atom of said amino acid derivative, wherein said reaction (A) is carried out simultaneously with a reaction of detaching said leaving group L from the β carbon atom of the amino acid derivative, and (B) a reaction of converting the amino group or carboxyl group bound to the α carbon atom of said amino acid derivative into a substituent to be the substrate for a hydrolase selective for D- or L-amino acids, and (II) reacting the amino acid derivative obtained in (I) with a hydrolase selective for either one of D- or L-amino acids, and subsequently separating the hydrolyzed D- or L-amino acid derivative. (I) carrying out the following reactions on an amino acid derivative to manufacture an amino acid derivative possessing a protected or non-protected thiol group at the β-position comprising D- and L-forms: (P) manufacturing an amino acid derivative possessing substituent R 1 and a leaving group L on the β carbon atom, wherein said step (P) comprises the following step (P-2): (P-2) reacting glycine with an aldehyde compound represented by R 1 CHO, (A) a reaction of introducing a protected or non-protected thiol group at the β carbon atom of said amino acid derivative, wherein said reaction (A) is carried out simultaneously with a reaction of detaching said leaving group L from the β carbon atom of the amino acid derivative, and (B) a reaction of converting the amino group or carboxyl group bound to the α carbon atom of said amino acid derivative into a substituent to be the substrate for a hydrolase selective for D- or L-amino acids, and (P) manufacturing an amino acid derivative possessing substituent R 1 and a leaving group L on the β carbon atom, wherein said step (P) comprises the following step (P-2): (P-2) reacting glycine with an aldehyde compound represented by R 1 CHO, (A) a reaction of introducing a protected or non-protected thiol group at the β carbon atom of said amino acid derivative, wherein said reaction (A) is carried out simultaneously with a reaction of detaching said leaving group L from the β carbon atom of the amino acid derivative, and (B) a reaction of converting the amino group or carboxyl group bound to the α carbon atom of said amino acid derivative into a substituent to be the substrate for a hydrolase selective for D- or L-amino acids, and (II) reacting the amino acid derivative obtained in (I) with a hydrolase selective for either one of D- or L-amino acids, and subsequently separating the hydrolyzed D- or L-amino acid derivative. <EOS>