Glioblastoma (GBM) is a primary malignant brain tumor that comprises approximately 57% of all gliomas and 48% of all primary malignant central nervous system (CNS) tumors. Despite the best standard therapies, glioblastoma survivors have a short survival time, about 24 months on average. A multi-omics approach can provide a promising way to systematically understand complex biology. However, few studies have utilized omics analysis to investigate the impact of anticancer drugs on GBM. The current project aimed to evaluate the impacts of the anticancer medications (paclitaxel 4.2 nM and etoposide 10 μg/mL) solely and in pairwise combination on the metabolic and proteomic signatures of the U373 cell line utilizing the accurate ultra-high-performance liquid chromatography-electrospray ionization quadrupole time-of-flight mass spectrometry (UHPLC-ESI-QTOF-MS) analytical technology. After applying One-way ANOVA, 94 significantly altered metabolites in U373 cells were observed. The produced metabolites were then analyzed utilizing t-tests, volcano plots, and enrichment analysis modules. Herein, the analysis revealed distinct metabolites to be significantly dysregulated (nutriacholic acid, L-phenylalanine, L-arginine, guanosine, ADP, hypoxanthine, and guanine), and to a lesser extent, mevalonic acid in paclitaxel and/or etoposide treated cells. Furthermore, both urea and citric acid cycles, and metabolism of polyamines and amino acids (aspartate, arginine, and proline) were significantly enriched. While for proteomics, a total of 12 proteins were significantly dysregulated among the groups. These findings can aid in identifying new biomarkers associated with the utilized drugs and creating a map for targeted therapy. MTA2, RPLP2, RPA2, COX5A, TRIM28, SRRM2, and RPA3 were shown to be significantly upregulated in the combination group when compared to the control. Moreover, in the combination group, cytosolic ribosome, mRNA splicing, cellular response to stress, and protein DNA complex were significantly altered in