ABSTRACT Introduction Chemotherapy has advanced well in the treatment of Non-Small Cell Lung Cancers (NSCLCs), but its effectiveness is limited by drug resistance. Unfortunately, cancer drug resistance becoming inevitable involving alterations in epigenetic regulation, molecular signalling, and metabolic rewiring. Thus necessitates development of novel and improved modalities to treat NSCLCs. Previously we identified that reduced glutamine dependence associated with drug resistance in leukaemia cells despite a lack of expression of Glutamate-ammonia ligase (GLUL). Here, we focused to investigate the involvement of GLUL knock down in lung cancer cells in mediating drug resistance mechanism. Material and methods We generated A549 GLUL knockdown stable cell lines by CRISPR-Cas9 method and were screened with FDA approved chemotherapeutic agents. Cell growth were analysed by MTS assay and colony formation were assessed by clonogenic assay; Proteins expression were detected by westernblotting. Targeted metabolomics were done using LC-MS with stable isotopes labelled intermediates to identify potential metabolic flux changes in GLUL knockdown cells. Results and discussions In vitro analyses of cell viability assay showed that GLUL knockdown in A549 cell line mediates resistance towards the treatment of chemotherapeutic drugs including Pazopanib, Imatinib, Dasatinib and Docetaxel. While, westernblotting analyses revealed that reduction in apoptotic marker PARP cleavage in knockdown cells in response to chemo drugs with respect to controls. Further, we also observed that GLUL knockdown activates pro-survival signalling mechanism i.e., PI3K-AKT pathway via increased phosphorylation of AKTser473 and S6 ribosomal proteinser235/236 in mediating resistance. Additionally, in clonogenic assay, cells were grown in the presence of different chemotherapeutic drugs, evidently GLUL knock down cells were able to form more colonies than control cells. Since GLUL activity potentially can be linked to central energy metabolism via glutaminolysis we probed the metabolic fluxes in glycolysis and TCA pathways by means of 13C labelled Glutamine, Glutamate and Glucose respectively and interestingly we didn’t observe any significant changes in metabolic flux linking the resistant phenotype to alterations in energy utilisation by the cells. Conclusion Our study shows that GLUL Knockdown could induce chemo-resistance, through modulating apoptotic and pro-survival signalling. Thus, GLUL may serve as a potential target for NSCLC tumour progression with therapeutic significance.