Antimicrobial resistance (AMR) is one of the most serious problems affecting public health and safety. When treated by antibiotics, bacteria usually experience changes in morphology that can lead to the development of AMR. In this work, we propose a strategy to study mutation in protein expression during morphological changes of bacteria under the impact of antibiotics. The study is focused on small proteins that can be detected by matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), which is nowadays widely used in hospitals for bacterial identification. We used a gradient microfluidic chip to observe the morphological changes of various bacteria under the impact of antibiotics. Differential peaks related to the morphological changes were first figured out by MALDI-TOF MS, then identified by considering the molecular weight of both candidate proteins and their tryptic digested peptides, and further validated by a liquid chromatography tandem mass spectrometry (LC-MS/MS) based label-free quantitative proteomic method. Specifically, carbapenem-resistant Klebsiella pneumoniae (CR-KP), super extended spectrum β-lactamases Escherichia coli, Vibrio parahemolyticus from South America prawns, and carbapenem-resistant Pseudomonas aeruginosa were used as model samples to illustrate the strategy. Eight proteins closely correlated with its morphological change were identified for CR-KP. Among the eight proteins, three, i.e., fimbrial subunit type 3, penicillin-binding protein activator LpoB, and 30S ribosomal protein S14, were further verified at the transcriptome level.