Keratin-associated proteins (KAPs) are important constituents of the wool cuticle, comprised of the endo-, exocuticle and a-layers, which contribute significantly to the fibre's molecular and mechanical characteristics. Relatively little is known about the distribution of specific KAPs across these layers, and correct protein identification of individual KAPs is difficult due to extensive homology and identity among individual KAPs. We here present evidence that, by specifically exploiting the high-cysteine content of KAPs in the wool cuticle, using 2-nitro-5-thiocyanobenzoic acid (NTCB) cleavage in combination with tryptic digestion, a larger number of KAPs can be identified than with standard trypsin-only digests. A total of 27 KAPs were identified, six of which could only be identified using NTCB. Furthermore, NTCB-mediated cleavage of cuticle proteins generated unique peptides critical for unambiguous identification of two KAPs, as well as significantly increasing the overall sequence coverage of most identified KAPs. Interestingly, some of the peptides found to be unique to particular KAPs could only be found in either the exo- or endocuticle. We conclude that for the analysis of high sulphur proteomes, specific targeting of cysteine residues using chemical agents such as NTCB can provide critical information for unambiguous protein identification.