The comprehensive determination of the absolute configuration, enantiomeric ratio, and total amount of standard amino acids by optical methods adaptable to high-throughput screening with modern plate readers has remained a major challenge to date. We now present a small-molecular probe that smoothly reacts with amino acids and biothiols in aqueous solution and thereby generates distinct chiroptical responses to accomplish this task. The achiral sensor is readily available, inexpensive, and suitable for chiroptical analysis of each of the 19 standard amino acids, biothiols, aliphatic, and aromatic amines and amino alcohols. The sensing method is operationally simple, and data collection and processing are straightforward. The utility and practicality of the assay are demonstrated with the accurate analysis of 10 aspartic acid samples covering a wide concentration range and largely varying enantiomeric compositions. Accurate er sensing of 85 scalemic samples of Pro, Met, Cys, Ala, methylpyrrolidine, 1-(2-naphthyl)amine, and mixtures thereof is also presented.