Circumventing or overwhelming the bacterial adaptation capabilities is key to combatting multidrug-resistant pathogens like Pseudomonas aeruginosa. We investigated the physiological stress exerted by approved antibiotics (ciprofloxacin, levofloxacin, rifampicin, gentamicin, tobramycin, azithromycin, tigecycline, polymyxin B, colistin, ceftazidime, meropenem, piperacillin/tazobactam), experimental antibiotics (CHIR-090) and NSAIDs (acetylsalicylic acid (aspirin), diclofenac, ibuprofen), and studied the bacterial response on the proteome level. Radioactive pulse-labeling of newly synthesized proteins followed by 2D-PAGE was used to monitor the acute response of P. aeruginosa to antibiotic treatment. Subsequently, marker proteins were excised from non-radioactive gels and identified by mass spectrometry. We generated a reference library of P. aeruginosa proteomic responses and implemented a mathematical comparison of the profiles. Proteomic signatures were derived for clinically relevant target areas.