Updated project metadata. Cottonseed meal (CSM) contains a high amount of underutilised proteins due to the presence of gossypol, a toxic compound unsuitable for human consumption. We designed a protein extract process to generate CSM protein isolate (CSMPI) with ultra-low gossypol content to unlock their potential. This process utilised alkaline-based protein extraction with gossypol removal through a multistep acidified organic solvent treatment. Overall, our method successfully yielded 22% of CSMPI from CSM and achieved a protein purity of 92.6%. The method has reduced the free and total gossypol content of the post-treated CSMPI to 4.8ppm and 147.2ppm, respectively, which is much lower than the limit set by US FDA. Proteomics analysis indicated that the gossypol re-moval treatment increased protein identification and enhanced the nutritional quality of the CSMPI due to increased branched-chain amino acids (BCAAs) content after treatment. Overall, our results help to navigate and direct the application of CSMPI towards nutritive human food application.