When abnormally expressed or controlled, kinase activity can cause cellular dysregulation and contribute to the onset of several diseases, including cancer. Based on the understanding of kinase malfunction, the discovery of small organic molecules to alter kinase function has culminated in the development of targeted cancer therapy. However, limited selectivity and the emergence of drug resistance remain fundamental challenges. Most known kinase inhibitors are Type I inhibitors, ATP-competitive compounds such as dasatinib that bind to the ATP binding site. Type II inhibitors are compounds which bind partially in the ATP binding site and extend into an adjacent allosteric site that is present only in the inactive kinase conformation. Compared to Type I inhibitors, Type II inhibitors have been shown to possess advantageous pharmacological properties. As such, many Type II inhibitors currently on the market, such as imatinib, are very effective anti-cancer drugs. Mutations resistant to Type I/II inhibitors are emerging at a rapid pace and often limit the success of targeted cancer therapies. At present, there are more than 50 mutation sites conferring different levels of imatinib resistance. Recently, a number of Type IV inhibitors that function via allosteric modulation have demonstrated promise towards addressing mutation dependent drug resistance. As such, the need to identify and develop reversible inhibitors that are resistant to such mutations and bind with a high affinity is the focus of many research projects. The binding affinity of Type I, II and IV Bcr-Abl kinase inhibitors with wild type and four mutant Bcr-Abl kinases (H396P, M351T, Q252H, and T315I) were measured using TruBind? Back-Scattering Interferometry (BSI). BSI successfully demonstrated facile determination of equilibrium dissociation constants (Kd) for all systems, with a high degree of concordance with competition assay derived IC50 results. These results indicate that BSI binding studies both class I, II, and IIV kinase inhibitors can easily be performed, allowing for confirmation of target engagement as well as direct binding assessment of type II and IV kinase inhibitors against inactive Bcr-Abl kinase. The latter makes BSI an attractive biophysical technique for the study of second and third generation kinase inhibitors to address the challenges of kinase inhibitor drug resistance.