Structured microbial colonies called biofilms are immersed in the extracellular matrix that produce independently and show enhanced resilience to environmental stressors and antimicrobial treatments. To create efficient management measures, it is essential to comprehend the intricate molecular processes behind biofilm creation, maintenance, and dissemination. Large-scale protein analysis, or proteomics, has become a potent tool in biofilm research, offering a profound understanding of microbial physiology, intercellular interaction, and adaptive responses. Quantitative techniques, including label-free quantification (LFQ), stable isotope labelling, tandem mass tagging (TMT), and mass spectrometry-based proteomics, particularly shotgun proteomics, have made identifying proteins specific to biofilms easier. These consist of extracellular polymeric substances (EPS)-associated proteins, quorum sensing molecules, stress-response factors, and regulatory proteins. Differential protein expression between planktonic and biofilm-associated cells may be clarified using comparative proteomics, which also reveals metabolic changes and mechanisms of antibiotic resistance. Proteomics is used in industrial, environmental, and therapeutic biofilm research. Proteomic research in medical microbiology helps find possible biomarkers and treatment targets for diseases linked to biofilms. Biofilm proteomics is used in industry to guide food processing and water system biofouling avoidance measures. Proteomic findings improve our knowledge of biofilm-mediated bioremediation in environmental biotechnology. Proteomics offers a thorough understanding of biofilm dynamics, opening the door for innovative intervention techniques to lessen biofilm-related difficulties in various domains. In the following chapter, we will outline discussions on proteomics in the research of biofilms.