ETS transcription factors from the ERG and ETV1/4/5 subfamilies are overexpressed in the majority of prostate cancer patients and contribute to disease progression. Here, we develop two in vitro assays for the interaction of ETS transcription factors with DNA that are amenable for high throughput screening. Using ETS1 as a model, these assays were applied to screen 110 compounds derived from a high-throughput virtual screen. We find that the use of lower affinity DNA-binding sequences, similar to those which ERG and ETV1 bind to in prostate cells, allowed for higher inhibition from many of these test compounds. Further pilot experiments demonstrated that the in vitro assays are robust for ERG, ETV1, and ETV5, three of the ETS transcription factors that are overexpressed in prostate cancer.. CC-BY 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.