iScience Article Metabolomics-based phenotypic screens for evaluation of drug synergy via direct-infusion mass spectrometry Primary Screening PEDS Synergy validation & Synergy mechanism sample HRMS nESI chip Bioluminescence screening Candidate combinations 2.5 th percentile Bliss index 0 Spheroids in 96-well DIMS Quantify drug synergy Bliss/Loewe models Apoptosis assay MFA Secondary Screening Data mining Metabolic reprogramming in vitro in vivo 3D model Drug library Candidate combinations + Xiyuan Lu, G. Lavender Hackman, Achinto Saha, ..., John DiGiovanni, Alessia Lodi, Stefano Tiziani alessialodi@utexas.edu (A.L.) tiziani@utexas.edu (S.T.) Highlights High-content metabolomics screening platform with stable isotope tracers was developed The platform allows for assignment to different library screens and in vitro models New algorithm is robust to apply on omics datasets for drug synergy evaluation Novel drug combination to treat prostate cancer was discovered and validated in vivo Lu et al., iScience 25, 104221 May 20, 2022 ª 2022 The Authors. https://doi.org/10.1016/ j.isci.2022.104221 ll OPEN ACCESS