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The permeability barrier of the outer membrane in Gram-negative bacteria possess an inherent defense towards antibiotics and is subject of study using multidisciplinary approaches and cutting-edge techniques. In this study, a medium-high throughput assay based on liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) was optimized and applied for comparing the degree of uptake of antibiotics with different modes of action into E. coli and P. aeruginosa. This method allowed the elucidation of time-course profiles of rapidly accumulated compounds and helped to differentiate accumulation profiles of nine antibiotics between the two Gram-negative species. The strain transferability of this assay allows the systematic assessment of the uptake of a broad range of compounds in different microorganisms. Apart from an increased impermeability, pathogenic bacteria quickly adapt metabolically to cope with a wide variety of environmental stresses, including antibiotic stress. Exposure to sub-lethal but constant concentrations of antibiotics in the environment plays an important role in enabling bacteria to make use of tolerance and resistance traits. In this study, the metabolic profile of wild type P. aeruginosa treated with different classes of antibiotics at sub-lethal concentrations showed important differences under a short exposure of two hours, and a long exposure of more than seven hours. P. aeruginosa maintained high levels of virulence-related metabolites, such as rhamnolipids, as a quick response to sudden antibiotic stress, indicating the readiness of bacteria to adapt quickly to environmental challenges. Fluoroquinolones, among the most potent antibiotics to date, are known to propitiate diverse bacterial responses, such as growth inhibition, biofilm production, and increased oxidative-stress response. However, these effects are associated to their potent activity and thought to be due to target interactions. In this study, two P. aeruginosa strains, one fluoroquinolone-susceptible with MIC of ...