The dissociation constant, Kd, is an important parameter for characterizing proteinprotein interaction affinities. SUMOylation is one of the important protein post-translational modifications and it involves a multi-step enzymatic cascade reaction, resulting in peptide activation and substrate conjugation. Multiple covalent and non-covalent proteinprotein interactions are involved in this cascade. Techniques involving Forster resonance energy transfer (FRET) have been widely used in biological studies in vitro and in vivo, and they are very powerful tools for elucidating protein interactions in many regulatory cascades. In our previous studies, we reported the attempt to develop a new method for the determination of the Kd by FRET assay using the interaction of SUMO1 and its E2 ligase, Ubc9 as a test system. However, the generality and specifications of this new method have not been fully determined. Here we report a systematic approach for determining the dissociation constant (Kd) in the SUMOylation cascade and for further sensitivity and accuracy testing by the FRET technology. From a FRET donor to acceptor concentration ratio range of 440, the Kds of SUMO1 and Ubc9 consistently agree well with values from surface plasmon resonance and isothermal titration calorimetry. These results demonstrate the high sensitivity and accuracy of the FRET-based Kd determination approach. This technology, therefore, can be used in general for proteinprotein interaction dissociation constant determination. Biotechnol. Bioeng. 2012; 109: 28752883. (c) 2012 Wiley Periodicals, Inc.