Background: Ewing’s Sarcoma (EWS) is an aggressive solid tumor of the bone and soft tissue affecting nearly 250 adolescents and young adults each year. Prior research has demonstrated sensitivity of cell lines bearing the pathognomonic EWS-FLI1 translocation to poly ADP ribose polymerase inhibitors (PARPi). Single agent PARPi is unlikely to provide clinical response without the addition of DNA damaging agents. We therefore examined the single agent cytotoxicity of olaparib, veliparib, and BMN 673 as well as the cytotoxicity obtained with the combination with either irinotecan (SN-38) or temozolomide (TMZ). Additionally we have created a unique, clinically relevant EWS orthotopic xenograft mouse model for in vivo evaluation. Methods: EWS cell lines were optimized for 7 day in vitrohigh throughput analysis. Individual cell lines were exposed to each PARPi in dose response and also in combination with SN-38 or temozolomide in dose response. Cell viability was measured after 7 days via CellTiter-Glo. A murine orthotopic xenograft was created by injecting tumor cells suspended in matrigel into the femur marrow cavity of CD-1 nude mice. Results: Single agent cytotoxicity was achieved with all agents, however dramatic variability was noted in drug concentrations required to attain cytotoxicity. The greatest cytotoxic effect was obtained with BMN 673 followed by olaparib and veliparib. When combined with SN-38 there was a dramatic decrease in EC50 in all lines tested with all PARPi tested. When combined with TMZ the effect was less dramatic and seen only in combination with BMN 673. Conclusions: The combination of irinotecan to BMN 673, olaparib and veliparib provides superior in vitro cytotoxicity when compared to PARPi alone. We now have a clinically relevant orthotopic murine xenograft to test the in vitro results. The full understanding of pharmacokinetics will be critical to help identify the best pairing of cytotoxic agents to move forward in our orthotopic xenograft model.