Abstract Background: Pharmacodynamics (PD) endpoint assays are important biomarker(s) for target therapy/personalized medicine in oncology drug discovery & development. Identification of the informative PD biomarkers/readouts can support dose selection in clinical trials, identify the patient subsets, explore mechanism of action, or differentiate compound(s) over others. Emerging evidence indicates that enhanced autophagy promotes tumor progression in established tumors, and inhibition of autophagy is recognized as a novel adjuvant cancer therapeutic strategy when combined with conventional chemotherapy for maximum therapeutic efficacy and overcoming chemoresistance. PI3K/mTOR inhibitors have been developed for cancer treatment and, when combined with an autophagy inhibitor, display synergistically improved efficacy. This synergy consequently has lead to multiple clinical programs to assess combination therapy regimes. Development of reliable high throughput PD readouts to measure autophagy modulation is critical for discovery of selective modulators, and/or identification of biomarkers either for patient selection or drug combination strategy for clinical development. Methods: Human U87MG glioblastoma and MCF7 breast cancer cells were treated with the PI3K/mTOR inhibitor Rapamycin with or without the autophagy inhibitor Hydroxychloroquine (HCQ). Sequestered LC3 (microtubule-associated protein1 light chain 3), which is recognized as an autophagy marker, was detected in cells by flow cytometry. 7-AAD/Annexin V staining were used for cell viability analysis. The efficacy of individual compounds and optimal combination doses were identified with a cell proliferation assay. Results: Our results indicated that autophagy was induced by Rapamycin treatment. Autophagy inhibitor Hydroxychloroquine exhibits dose dependent inhibition of in vitro proliferation of U87 MG and MCF-7 tumor cells. U87 MG cells are more sensitive to autophagy inhibition than MCF-7 breast cancer cells. Enhanced inhibition of cellular proliferation and increased cell death were observed in U87MG and MCF-7 cells in combination regime when compared to treatment with either compound alone. Conclusion: We developed a High Throughput flow cytometry based method for screening pharmacodynamics biomarker panel for supporting dose selection and/or identify optimal combinations in oncology drug discovery& development. The dynamic impacts of treatments on the activation status of autophagy at various time points are currently under investigation, and we are continuing to expand flow cytometry panels by incorporating other potential biomarkers. Studies are funded by EMD Millipore (a division of Merck KGaA, Darmstadt, Germany). Note: This abstract was not presented at the meeting. Citation Format: Zheng Feng, Theodore Baginski, Michael Donio, Diane Werth, Jacob Bode. Development of high throughput flow cytometry assays to support target therapy/personalized medicine in oncology drug discovery and development: Developing and optimizing a flow cytometry-based autophagy detection method/panel for oncology drug discovery and development. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 323. doi:10.1158/1538-7445.AM2014-323