The ability to selectively conduct K+ ions is central to the function of K+ channels. Selection for K+ and rejection of Na+ takes place in a conserved structural element referred to as the selectivity filter. The selectivity filter consists of four K+-specific ion binding sites that are created using predominantly the backbone carbonyl oxygen atoms. Due to the involvement of the protein backbone, experimental manipulation of the ion binding sites in the selectivity filter is not possible using traditional site directed mutagenesis. The limited suitability of the site-directed mutagenesis for studies on the selectivity filter has motivated the development of a semisynthesis approach, which enables the use of chemical synthesis to manipulate the selectivity filter. In this chapter, we describe the protocols that are presently used in our laboratory for the semisynthesis of the bacterial K+ channel, KcsA. We show the introduction of a spectroscopic probe into the KcsA channel using semisynthesis. We also review previous applications of semisynthesis in investigations of K+ channels. While the protocols described in this chapter are for the KcsA K+ channel, we anticipate that similar protocols will also be applicable for the semisynthesis of other integral membrane proteins.