Publisher Summary This chapter discusses the estimation of phosphorylation stoichiometry by separation of phosphorylated isoforms. The stoichiometry of phosphorylation of a protein is of central importance in assessing the possible significance of the phosphorylation. Although it is theoretically possible that enzymatic function could be regulated by the rate of phosphorylation or rate of dephosphorylation of an enzyme, generally it is the phosphorylation state that determines activity. The actual rates of phosphate esterification and hydrolysis are important as their balance determines the extent of phosphorylation of the substrate. There are two primary ways of assessing phosphorylation stoichiometry—radioactive method and physically separating the phosphoprotein and dephosphoprotein and then estimating their relative quantities. .