The proteins in human hair consist primarily of microfibrillar keratins with a molecular mass of 40–65 kDa and keratin-associated proteins (KAPs) with a molecular mass of 6–30 kDa, according to the results obtained from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Because an effective purification procedure of KAPs has not been established, little is known about the protein chemistry of KAPs as compared with that of keratin. When hair samples were incubated in the Shindai solution containing alcohols such as methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, and 2-methyl-1-propanol, the extraction of KAPs was enhanced, while extraction of keratin was suppressed. Using ethanol, we established a selective purification procedure for KAPs and keratin. According to Tricine/SDS-PAGE, the KAPs fraction contained six polypeptides with molecular masses of 3.5, 4.4, 5.2, 7.8, 15, and 28 kDa. The keratin fraction contained two polypeptides with molecular masses of 45 and 67 kDa and was free of low-molecular-weight components. The amino acid compositions of the KAPs and keratin fractions were mostly in agreement with the values found in the literature. The recoveries of the KAPs and keratin fractions from the hair samples were approximately 10 and 50%, respectively. Scanning electron microscopy (SEM) showed that hair samples retained fine fibrous structures in the cortex after extracting the KAPs and that the additional extraction of keratin caused the fibrous structures to disappear. These results indicated that KAPs may function by surrounding the fibrous structures and supporting the keratin fibers in the cortex. In this study, we propose a novel and convenient isolation procedure for KAPs and keratin from human hair.