This chapter discusses chromatography, which is a versatile and powerful tool for the separation and quantitation of a wide variety of physiologically important substances. The primary goal of the chromatographic process is to separate and resolve a mixture of substances into individual components which are called solutes. Paper chromatography of amino acids is the most widely used procedure for separation and identification of amino acids, which uses filter paper as the supporting medium. A strip of filter paper is suspended vertically with the lower end dipping into a mixture of water and some organic solvent. Water, which is held strongly by the paper because of its affinity for the material, represents the stationary phase. Rf is the ratio of distance travelled by the solute to distance travelled by the solvent. In a given solvent system, each amino acid has a characteristic Rf value which can be used to identify it.