Peptidomics is the comprehensive study of endogenous peptides, is a powerful analytical tool providing deep insights into biological systems. This chapter explores the fundamentals of peptidomics, from its core methodologies centered on mass spectrometry and bioinformatics to its diverse applications. While crucial in biomedical research for biomarker discovery, its impact on aquaculture and fish biology is particularly significant. Peptidomics is revolutionizing the sector by enabling the valorization of fish processing by-products into high-value bioactive peptides with antioxidant and antihypertensive properties. It is also instrumental in discovering novel antimicrobial peptides (AMPs) to combat disease, identifying stress-responsive biomarkers for monitoring fish welfare, and ensuring seafood quality. In spite of challenges like a limited database, integration of peptidomics with multi-omics technologies promises to foster a more sustainable and efficient aquaculture industry.