Antimicrobial resistance (AMR) poses significant challenges to global public health. The major cause of AMR development is previous use of antibiotics, hospitalization, and the lack of efficient methods for screening AMR pathogens. Mass spectrometry techniques offer rapid, sensitive, and early detection of AMR both on proteomics and metabolomics levels. Hence, a metabolomics analysis on clinical isolates of A. baumannii was conducted to understand the resistance patterns exhibited by these isolates. A. baumannii (ATCC strain) and its clinical isolates (n = 26) were screened against five antibiotics i.e., ciprofloxacin, colistin, cefixime, gentamicin, and co-amoxiclav to obtain their resistance profile using antibiogram and MIC methods. After that, all the samples were analyzed in both positive and negative modes of Flow Injection-High Resolution-Electrospray Ionization Mass Spectrometry (FI-HR-ESI-MS) after 6 h of incubation. Data analysis revealed the identification of a total of 43 metabolites. The metabolites were then subjected to chemometric analysis to find any significant association of the metabolites with an increase in the MIC values. The chemometric analysis resulted in a total of eleven metabolites with p-values<
0.05 to be significantly associated with the resistance of A. baumannii isolates against the drugs. The concentrations of two metabolites, pyochelin, and L-serine, increased sequentially with the increase in MIC values (increase in resistance) of ciprofloxacin and cefixime, respectively. The study showed a significant association of metabolites with the resistance in A. baumannii isolates and can play a potential role in the development of new therapeutics against the arising antimicrobial resistance of A. baumannii towards various antibiotic drugs.