Amino acids involved in cottonseed protein cross-linking by formaldehyde, glutaraldehyde, and glyoxal during protein film formation were identified by an original technique. The entire HPLC amino acid profile (after acid hydrolysis) was studied, along with variations in reactive lysine contents, in films cross-linked or not with increasing quantities of formaldehyde, glutaraldehyde, and glyoxal. This strategy highlighted the formation of acid-resistant lysine derivatives that a simple reactive lysine determination would not have detected. The results-which agree with previously published data-enhance the overall understanding of cross-linking activities that occur in aqueous alkaline solutions during the formation of protein films made with cottonseed flour. Lysine was found to have a key role in protein cross-linking by dialdehydes, with the involvement of tyrosine in the presence of formaldehyde and of arginine in the presence of glyoxal. These results could provide valuable chemical tools for adjusting the mechanical properties of cottonseed protein films.