Abstract It has been found that erythro-β-hydroxy-Dl-norvaline, and erythro-β-hydroxy-Dl-norleucine, and threo-β-hydroxy-Dl-leucine form scarcely-soluble compounds in water with α-naphthylphosphoric acid, tetrachlorophthalic acid, and chlorendic acid, respectively. On the other hand, the compounds of their diastereoisomers with the reagents were soluble in water. On the basis of these facts, β-hydroxy-Dl-norvaline, β-hydroxy-Dl-norleucine, and β-hydroxy-Dl-leucine were successfully separated into their diastereoisomers. The configurational assignment of α-amino-β-hydroxy acids without a branched side chain on the γ-carbon atom was established by means of NMR spectroscopy.