Shaker-type voltage-gated K⁺ (Kv1.x) channels are composed of a tetramer of pore-forming α subunits which can associate with regulatory proteins such as the Kvβ subunits to form an α4β4 complex. Cytosolic Kvβ subunits, which interact with Kvα subunits through binding to the T1 domain, have been demonstrated to modify the kinetics of Kv channels. Thus understanding Kvα-β interactions, may offer a novel mechanism to exploit the function of Kv channels for therapeutic benefit. The aim of this study was to develop a robust and sensitive assay to determine the nature of the interaction between rKvα1.1 T1 domains and rKv β1 core proteins to allow subsequent identification of compounds which modulate this interaction. rKvα 1.1 T1 domains and rKv β1 core proteins were expressed in E. coli as specific fusion proteins, incorporating biotin carboxyl carrier protein as an affinity tag to the rKv β1 core protein. Purified rKvα 1.1 T1 domains were labelled with Cy3 dye. rKv β1 core proteins were immobilised through biotin-streptavidin interactions in a microwell format, with rKvα 1.1 T1 domains titrated into the microwells to form the basis of a fluorescent-readout protein-protein interaction assay. In order to optimise the assay, binding kinetics of the interaction between rKvα 1.1 T1 domains and rKv β1 core proteins were defined (analyses derived from Davies et al. 1999), see Table 1. The characterisation of rKvα 1.1 T1 domain – rKv β1 core protein interactions enabled construction of a cell-free displacement assay based in a microplate format. Assay parameters were defined following optimisation of the assay for a 96 well plate format. Throughput in this semi-automated process is 200 compounds per day with assay plate coefficient of variation less than 10% (n > 20). A compound series was identified that modulated the interaction between rKv α 1.1 T1 domains and rKv β1 core proteins (see Table 2 for the compound series). In summary, determination of the binding kinetics of the interaction between rKv α1.1 T1 domains and rKv β1 core proteins allowed the construction of a robust, fully scaleable assay that was used to identify compounds that modulate this interaction. Compounds identified within this assay may serve to modulate Kv channel kinetics through a novel mode of action.