With the recent interest in targeting the DNA damage response (DDR) and DNA repair, new screening methodologies are needed to broaden the scope of targetable proteins beyond kinases and traditional enzymes. Many of the proteins involved in the DDR and repair impart their activity by making specific contacts with DNA. These protein–nucleic acid interactions represent a tractable target for perturbation with small molecules. We describe a high throughput, solution-based equilibrium binding fluorescence polarization assay that can be applied to a wide array of protein–nucleic acid interactions. The assay is sensitive, stable, and able to identify small molecules capable of blocking DNA–protein interactions.