The structure of protein kinases has been extensively studied using protein crystallography. Conformational movement of the kinase activation loop is thought to be crucial for regulation of activity, however, in many cases the position of the activation loop in solution is unknown. Protein kinases are an important class of therapeutic target and kinase inhibitors are classified by their effect on the activation loop. Here, we report the use of pulsed electron double resonance (PELDOR) and site directed spin-labeling to monitor conformational changes through the insertion of MTSL on the dynamic activation loop and a stable site on the outer surface of the enzyme. The action of different ligands such as microtubule-associated protein (TPX2) and inhibitors could be discriminated as well as their ability to lock the activation loop in a fixed conformation. The study provided evidence for structural adaptations which could be used for drug design and a methodological approach which has potential to characterize inhibitors in development.