A sensitive and specific assay method for cysteine sulfinic acid (CSA) and cysteic acid (CA) using high-performance liquid chromatography has been developed. The method includes post-column derivatization of various amino acids with o-phthalaldehyde in the presence of 2-mercaptoethanol. The column packed with cation-exchange resin (ISC-07/S1504, Shimadzu Scientific Instruments, Inc., Kyoto, Japan) was used for obtaining general separation of amino acids except CSA and CA, while the separation of CSA and CA was achieved using a strong-base anion exchange (ISA-07/S2504, Shimadzu Scientific Instruments) column. The fluorescence peak area for CSA was linear between 20 pmol and 5 nmol, whereas that for CA was 10 pmol to 5 nmol. The regional distribution of CSA, CA, and other amino acids in the rat brain was studied using this new assay method.