Quantifying the binding energy in DNA-protein interactions is of critical importance to understand transcriptional regulation. Based on a simple computational model, this study describes a high-throughput percentage-of-binding strategy to measure the binding energy in DNA-protein interactions between the Shewanella oneidensis ArcA two-component transcription factor protein and a systematic set of mutants in an ArcA-P (phosphorylated ArcA) binding site. The binding energies corresponding to each of the 4 nt at each position in the 15-bp binding site were used to construct a position-specific energy matrix (PEM) that allowed a reliable prediction of ArcA-P binding sites not only in Shewanella but also in related bacterial genomes.