Keratin, as one of the most abundant and underexploited protein sources, is a ubiquitous biological material that commonly exists in epithelial cells. Due to the excellent biocompatibility and biodegradability, keratin is widely used in biomedical applications. Previously, these biomaterials were prepared by dissolving and extracting the keratinous materials. However, the keratins obtained by direct extraction is not pure and contain many by-products. Moreover, natural keratins suffer from limited sequence tenability. In comparison, the recombinant keratin proteins produced by recombinant technology can overcome these drawbacks while maintaining the desired chemical and physical characteristics of natural keratins. Accordingly, this chapter mainly introduces the experimental protocols of the recombination of keratin. As these recombinant keratins are often used for assembly of intermediate filaments (IFs) in vitro, assembly protocols are also introduced in this chapter.