Abstract 3D tumor organoids are pivotal tools to study tumor development, propagation and invasive properties (Tanner and Gottesman, 2015). They closely recapitulate many characteristics of the tumor of origin in terms of structure, heterogeneity, complexity and response to drug treatment. As such, they are considered an accurate pre-clinical model for drug discovery and development (Nyga et al, 2011; Tanner and Gottesman, 2015; Shroyer, 2016). Nevertheless, reliable methods to perform high-throughput drug screenings using tumor organoids are limited and require extensive manipulation such as sample transfer from plate to plate. In addition, thick 3D matrix supports may hinder drug penetration. Here we introduce a robust method to generate, treat and assay 3D tumor organoids in standard, uncoated 96- or 384-well plates. By plating cells around the rim of the wells we generate an ultrathin layer of Matrigel that allows penetration of small molecules and bulkier peptide drugs. Media change is easily performed from the center of the well without perturbing the organoids. After a 2-day treatment course, organoids are released by dispase treatment and assayed in the same well by performing a CellTiter-Glo assay (Promega). The method exhibits low plate-to-plate, experiment-to-experiment and operator-to-operator variability. In addition, it can be extended to patient-derived tumor organoids (PDTO) generated from primary tumors. We show how PDTO from high-grade serous ovarian carcinoma (HGSOC) specimens are generated with a success rate >95%. Given the low number of cells required and high assay sensitivity, we can seed 200 wells from 1Mio cells derived from a tumor biopsy, and test over 60 different drugs/concentrations in triplicates. Moreover, we show how drug combination studies are feasible and allow assessing treatment synergies. As an example, we combined a peptidic inhibitor of p53 aggregation, ReACp53 (Soragni et al, 2016), with carboplatin in PDTO HGSOC established from a panel of patients harboring different p53 mutations. Drug interaction studies are performed across a range of ReACp53 and carboplatin concentrations and cell viability assessed at day 2 is used to compute drug-response curves, EC50 values and combination indexes. In conclusion, we are introducing an inexpensive, robust, easily automated method to perform high-throughput drug screenings in 3D organoids and PDTO and demonstrate its utility in determining drug sensitivities and optimizing multi-drug combinations in vitro. Citation Format: Nhan Phan, Jessica Huang, David Eisenberg, Sanaz Memarzadeh, Alice Soragni. A novel method for high-throughput drug screening in 3D tumor organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5782. doi:10.1158/1538-7445.AM2017-5782