Filamentation is one of the self-saving responses and a survival strategy of bacteria in the circumstance of antibiotics. Understanding the molecular mechanism of filamentation can lead to the development of new inhibition methods for antibiotic-resistant bacteria. In this work, we develop an online microfluidics mass spectrometry for real‐time analysis of metabolic changes of bacteria during filamentation under the stimulus of antibiotics. Together with untargeted proteomic analyses and fluorescence imaging of target enzymes, significant pathways, e.g. nucleotide metabolism and coenzyme A biosynthesis, correlated to the filamentation of extended spectrum beta‐lactamase producing E. coli (ESBL-E. coli) are identified.