In aquaculture, proteomics has emerged as a powerful tool that offers deep insights into protein functions and their expression patterns in response to environmental stressors, diet, disease etc. Such knowledge ensures sustainability in aquaculture production and enhances food security for billions of populations. This chapter focuses on gel-based and gel-free proteomics techniques and advanced mass spectrometry, which provide insights into protein changes in varying aquaculture conditions. Proteomic techniques offer valuable information on protein biomarkers indicating fish health, welfare, immune responses, and adaptive mechanisms to environmental challenges. Of the proteins of interest identified, these include heat shock proteins (HSPs), some antioxidant defence enzymes linked to stress response, and others related to nutritional adequacy and disease resilience. Proteomics thus opens the way for better dietary formulation, selective breeding, disease diagnostics, and vaccine development to ensure better fish welfare and reduce antibiotic dependency. Proteomics also helps in assessing environmental impacts and practice optimization under fluctuating conditions to maintain fish health. Aquaculture can maintain a holistic understanding of fish biology by integrating proteomics with other “omics” technologies to promote efficient and ethical aquaculture practices vital for global food security.