Antibiotic resistance is a growing public health crisis that occurs when bacteria evolve and become resistant to the drugs designed to kill them. As a consequence, common infections can become harder to treat, leading to increased morbidity, mortality, and healthcare costs. Analyzing the metabolic activities of microorganisms is essential in combating antibiotic resistance. By understanding how bacteria adapt their metabolism to antibiotics, researchers can develop more eÉective treatment strategies, ultimately reducing the impact of resistant infections on public health. Proteus mirabilis is a critical opportunistic pathogen associated with urinary tract infections, especially in women. The rise of resistant strains complicates treatment and poses a serious challenge in clinical settings. This study aims to investigate the development of antibiotic resistance in P. mirabilis when exposed to sub-inhibitory concentrations of ampicillin and to explore whether such resistance leads to cross-resistance to other antibiotics. Using the disk diÉusion method, resistant strains were generated through gradual exposure to sub-inhibitory ampicillin concentrations. It was found that ampicillin-resistant strains exhibited cross-resistance to ceftazidime. Subsequently, it was compared the metabolomic profiles of these resistant strains with sensitive control strains. For metabolic profiling, each sample was analyzed by GC-MS. The metabolomic data, supported by statistical and bioinformatic analyses, enhance our understanding of metabolic pathways, elucidate metabolic changes, and clarify interactions among metabolites. It was concluded that during the development of antibiotic resistance, P. mirabilis passages exhibited significant alterations in vitamin B6 metabolism, unsaturated fatty acid biosynthesis, and tyrosine metabolism pathways. These pathways interact to regulate vitamin B6 metabolism and support the survival and resistance of P. mirabilis in response to antibiotics.