Sequence features, including the binding affinity of binding motifs for their cognate transcription factors, are important contributors to promoter behavior. The ability to predictably recode binding affinity enables the development of synthetic promoters with varying levels of response to known cellular signals. Here we describe a luminescence-based microplate assay for quantifying the relative affinity of transcription factors for short DNA probes. We then demonstrate how relative binding affinity data can be used to design synthetic plant promoters of varying strengths that respond to the same transcription factor.