Bioactive molecules (or signaling molecules) are involved in tissue regeneration and have an important role in modulating the microenvironment in vivo. Bioactive chemicals have the ability to govern host cell motility, proliferation, and differentiation, as well as enabling cells to interact with their surrounding microenvironment via particular receptors for chemical recognition. This chapter introduces ion-exchange chromatography (IEC) as a method for separating and purifying bioactive compounds such as polyphenols, catechin derivatives from complicated plant mixtures, proteins, minor whey protein, peptides, human C-peptide, alkaloids from Chinese medicines, plasmid DNA and carbohydrates.