The misuse of antimicrobials has rapidly spread antimicrobial resistance (AMR) in microorganisms, posing a global health threat to eÉective infection treatments. Understanding how microorganisms respond to antibiotics is crucial for developing new strategies and drug discovery. In this study, E. coli strains with diÉerent resistance profiles against gentamicin, which is an aminoglycoside antibiotic that is primarily used to treat serious infections caused by Gram- bacteria, were examined by MS based proteomics methods to understand the changes induced by the antibiotic. Proteomic analysis of E. coli strains was performed using a UPLC connected to timsTOF-MS/MS system mass spectrometry followed by a data processing step using MaxQuant and Perseus. In total, 2080 proteins were identified and 340 proteins were found to be significant. The diÉerentially expressed proteins were classified by Gene Ontology analysis based on their location, biological processes, and molecular functions. It was observed that the majority of these proteins play a key role in intracellular anatomical structures, including the cytoplasm, membrane and cytosol, which are crucial for maintaining cellular organization, facilitating transport, and enabling communication between cellular compartments. Notably, gentamicin was found to impact primary cellular processes, including the cell cycle, cell division, and metabolic regulation, particularly influencing protein synthesis and the cellular response to stress. This suggests gentamicin not only alters protein expression but may also disrupt essential cellular functions, potentially causing to cytotoxic eÉects. In terms of molecular functions, the analysis revealed that the diÉerentially expressed proteins possess catalytic activity, highlighting their roles as enzymes that facilitate biochemical reactions within the cell. This implies that many of the aÉected proteins are vital for metabolic pathways and play a significant role in the overall cellular response to gentamicin. Furthermore, some proteins showed binding capabilities, including nucleic acid, ion, and lipid binding, highlighting their diverse functions in cellular processes.