High‐throughput screening was used to isolate synergistic compounds from a library of 136 natural products that exhibited negligible effects on non‐malignant cell lines. Three groups were screened including four primary cell lines isolated from pediatric patients with pre‐B ALL (acute lymphoblastic leukemia), five pediatric immortalized B‐ALL cell lines, and three non‐malignant cell lines. A total of 9 compounds were selected from the primary round of screening, these exhibited an inhibition average of at least 20% in the primary B‐ALL cell lines with a consistent trend in the immortalized B‐ALL cell lines. Of these compounds, parthenolide had the most negligible effect on the non‐malignant cell lines, the highest efficacy across the all B‐ALL cell lines, and was therefore selected as the top candidate. The remaining treatments were grouped as toxic or selective depending on if the average inhibition of the non‐malignant cell lines was greater than 50%. A total of 42 combinations were evaluated for each treatment pair in the second round of screening; the Chou‐Talalay and BLISS models were utilized to determine synergism. Based on both models, parthenolide was observed to have the highest and most consistent synergistic relationship with shikonin across all pre‐B ALL cell lines while showing no toxicity in the non‐malignant cell lines. Untargeted metabolomics and metabolic flux analysis using isotopically labeled glutamine and glucose was performed to analyze the impact of the combinatorial treatment on pediatric pre‐B cell acute lymphoblastic leukemia metabolism.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.