Surface plasmon resonance (SPR) has been used extensively in the field of DNA/DNA, DNA/protein, and small molecule protein/DNA interactions. However, there have been growing concerns with regard to the proper designing of experiments and the quality of analysis and reporting of SPR results. Here we describe a protocol that is designed to address some of those issues. It encompasses procedural steps beginning with immobilization of streptavidin on CM5 chips to the final step of data reporting on DNA-polymerase interaction binding kinetics. In evaluating the protocol, we carried out experiments using a simple methodology developed in our laboratory, taking advantage of the high sensitivity and superior signal-to-noise ratio of Biacore T200. We probed the binary and ternary binding affinities between exonuclease-deficient Klenow fragment (Kf-exo-) and various arylamine DNA lesions. We employed unmodified and carcinogen-modified oligonucleotides in the presence and absence of dNTPs. The total time required to carry out the method to completion is between one and two weeks, approximately two days for the SPR binding assays and one week for synthesis, purification, and characterization of modified oligonucleotides. Though the protocol presented here is meant for Biacore T100 or T200 model, the overall methodology can be applied for other instruments also.