Abstract Soft tissue sarcomas (STS) are a highly heterogeneous collection of tumors that arise from primitive mesenchymal cells. Due to a reliance on cytotoxic chemotherapy as standard of care, survival outcomes for advanced STS patients remain poor. While efforts to understand sarcomagenesis have revealed potential biomarkers, the diversity of STS subtypes and interpatient tumour heterogeneity has led to the lack of effective molecular targeted therapies available. Given the uniquely individualized nature of STS, we hypothesize that the application of an experimental-analytics method, Quadratic Phenotypic Optimization Platform (QPOP), towards primary STS patient samples can improve identification of patient-specific drug combinations. Across a total of 45 patient samples collected, QPOP identified a novel drug pair as most frequently top-ranked, outperforming standard of care ifosfamide and doxorubicin. Interestingly, analysis of QPOP ranking trends revealed this combination to be broadly effective, implying common targetable pathways independent of STS subtype. Single-drug and combination dose-response assays performed in a panel of established patient lines supported its synergistic interaction, accompanied by increased PARP and caspase-3 cleavage with immunoblotting, and increased apoptotic response with Annexin V assay. Mechanistically, this synergy results in cell cycle arrest and induction of programmed cell death pathways, as revealed by molecular profiling on representative patient and cell lines. We further showed that co-treatment synergistically repressed oncogenic MYC and activated the p27kip1 tumor suppressor. Finally, this combination was more effective at impairing tumor progression than single agents alone in an STS xenograft mouse model. Overall, work here provides preliminary clinical evidence for the prioritization of a novel drug pair in addressing STS beyond known clinicopathologic parameters. Citation Format: Sharon Pei Yi Chan, Wai Yee Wong, Nur Nadiah Ismail, Baiwen Luo, Benjamin Jieming Chen, Brandon Xuan Ming Phua, Xing Yi Woo, Tan Boon Toh, Edward Kai-Hua Chow, Valerie Shiwen Yang. Ex vivo Drug Testing Identifies a Novel Drug Pair in Targeting STS abstract. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P41.