Abstract Most current therapeutic approaches to cancer include agents which induce genotoxic damage, either through direct damaging effects on DNA or indirectly, by altering cellular checkpoint controls or by causing a synthetic lethal response in patients with genetic abnormalities in DNA damage repair pathways. As candidate agents or novel combination therapies come into the clinic, the development of multiplexed immunoassays that quantitatively measure several pharmacodynamic biomarkers of DNA repair could improve our understanding of the relationship between target engagement and therapeutic efficacy. We previously reported the initial development of a quantitative, multiplex immunofluorescence assay comprised of a nuclear marker and four DNA damage repair proteins (γH2AX, pNbs1S343, Rad51, and ERCC1) as a method for monitoring drug action in vitro and in vivo. Here, we report the use of this multiplex assay to measure the effect of a PARP inhibitor (BMN-673) on an isogenic pair of HR competent and incompetent cell lines. The multiplex immunofluorescence assay utilizes a cassette approach, with all validated primary antibodies conjugated directly to a fluorophore or hapten such that multiple antibodies raised in the same host species can be used without the requirement of an anti-species reporter. The cassette approach allows for rearrangement of the composition of markers in the multiplex assay based on the investigational agent or clinical trial in question. We will also report on the development and utility of additional DNA damage markers for future multiplex assays, including markers specific for ATR-activated repair pathways. We have developed an antibody to the pATR T1989 site and will demonstrate its use in immunofluorescence applications as a pharmacodyamic marker following in vitro exposure to DNA damage-inducing agents and an ATR inhibitor. Additionally, we show in vitro data which supports the use of phosphorylated checkpoint proteins (pChk1S345, pChk2T68) as downstream indicators of DNA damage in the multiplex format. These biomarkers, in conjunction with our previously-reported multiplex panel, will provide an effective technique to monitor DNA repair responses and checkpoint controls in the clinical evaluation of combination therapy approaches targeting DNA damage repair deficiencies. Funded by NCI Contract No. HHSN261200800001E. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):PR11. Citation Format: Allison M. Marrero, Deborah Wilsker, Scott M. Lawrence, Thomas D. Pfister, John Hirt, Ralph E. Parchment, Joseph E. Tomaszewski, James H. Doroshow, Robert J. Kinders. Use of a DNA damage multiplex immunofluorescence assay to monitor pharmacodynamic markers following in vitro exposure to therapeutic agents. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr PR11.