We have developed a novel framework that can be applied for the analysis of signal transduction networks, both to facilitate reconstruction of the network structure and quantitatively characterize the interaction between network components. This approach, termed activation ratio analysis, involves the ratio between active and inactive forms of signaling intermediates at steady state. The activation ratio of an intermediate is shown to depend linearly upon the concentration of the activating enzyme. The slope of the line is defined as the activation factor, and is determined by the kinetic parameters of activation and inactivation. When activation ratios for simple signaling systems are considered, a set of rules develop that can be used to transform a set of experimental data to a proposed model network structure, with activation factors yielding a measure of activation potential between intermediates. / Singapore-MIT Alliance (SMA)