This study investigated some physicochemical properties of keratin extracted from Merino wool using five chemical extraction methods: alkali hydrolysis, sulfitolysis, reduction, oxidation, and extraction using ionic liquid. The ionic liquid method produced the highest protein yield (95%), followed by sulfitolysis method (89%), while the highest extraction yield was obtained with the reduction method (54%). The lowest yield was obtained with the oxidation method (6%). The oxidation extract contained higher molecular weight (>40 kDa) protein components, whereas the alkali hydrolysis extract contained protein material of <10 kDa. The sulfitolysis, reduction, and ionic liquid extracts contained various protein components between 3.5 and 60 kDa. Keratin obtained from various extraction methods had different yield, morphology, and physicochemical properties. None of the samples were toxic to L929 fibroblast cells up to a concentration of 2.5 mg/mL. Apart from the alkali hydrolysis extract, all other keratin extracts (reduction, sulfitolysis, ionic liquid, and oxidation) showed Fourier transform infrared adsorption peaks attributed to the sulfitolysis–oxidation stretching vibrations of cysteine-S-sulfonated residues, with the oxidation extract showing the highest content of cysteine-S-sulfonated residues. This study indicates that the properties of the keratin extract obtained vary depending on the extraction method used, which has implications for use in structural biomaterial applications.