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Glutamate-ammonia ligase (GLUL) is important for acid-base homeostasis, ammonia detoxification, cell signaling, and proliferation. Here, we reported that GLUL ablation conferred resistance to several anticancer drugs in specific cancer cell lines while leaving other cell lines non-resistant to the same drugs. To understand the biochemical mechanics supporting this drug resistance, we compared drug-resistant GLUL knockout (KO) A549 non-small-cell lung carcinoma (NSCLC) cells with non-resistant GLUL KO H1299 NSCLC cells and found that the resistant A549 cells, to a larger extent, depended on exogenous glucose for proliferation. As GLUL activity is linked to the tricarboxylic acid (TCA) cycle via reversed glutaminolysis, we probed carbon flux through both glycolysis and TCA pathways by means of ¹³C5 glutamine, ¹³C5 glutamate, and ¹³C6 glucose tracing. We observed increased labeling of malate and aspartate in A549 GLUL KO cells, whereas the non-resistant GLUL KO H1299 cells displayed decreased ¹³C-labeling. The malate and aspartate shuttle supported cellular NADH production and was associated with cellular metabolic fitness. Inhibition of the malate-aspartate shuttle with aminooxyacetic acid significantly impacted upon cell viability with an IC₅₀ of 11.5 μM in resistant GLUL KO A549 cells compared to 28 μM in control A549 cells, linking resistance to the malate-aspartate shuttle. Additionally, rescuing GLUL expression in A549 KO cells increased drug sensitivity. We proposed a novel metabolic mechanism in cancer drug resistance where the increased capacity of the malate-aspartate shuttle increased metabolic fitness, thereby facilitating cancer cells to escape drug pressure.