Using gene fusion technology, polypeptide fusion tags can be engineered into target proteins at the genetic level. The resultant recombinant proteins may possess the biochemical properties of the imported fusion tags. Therefore, it is possible to take advantage of fusion tags to improve and evaluate protein expression, to detect and track protein targets, and to purify and characterize proteins. However, it is necessary to eliminate any influence of the fusion tag in structural characterization experiments or in isolating pharmaceutical proteins. Scientists must therefore remove fusion tags prior to structural and functional analyses when fusion tags are suspected of interfering with the biological activity of a protein or influencing its behavior. The fusion tag can be removed by several methods including harsh chemical treatment, mild enzymatic cleavage by endoprotease or exoprotease, and intein-mediated self-cleavage. Here the literature is reviewed in relation to principles, applications, and approaches of each method.