Innate immunity serves as the primary defense system in fish, offering rapid and nonspecific protection against pathogens. This chapter offers a comprehensive review of key methodologies used to evaluate innate immune responses in fish, which is essential for understanding their health status. The introduction highlights the significance of innate immunity in fish and its relevance to aquaculture and fisheries research. Detailed descriptions of various assays, including their principles, reagents, and procedures, are provided. The serum bactericidal and serum antiprotease assays assess proteolytic enzyme inhibition, while the serum myeloperoxidase (MPO) and serum lysozyme activity assays evaluate enzymes critical for microbial killing. The alternative complement pathway assay measures complement activation, a vital aspect of innate immunity. Cellular immune responses are explored through assays that assess leukocyte functions, including the measurement of superoxide anion and nitric oxide production, which indicate oxidative and nitrosative responses. Intracellular MPO activity in leukocytes highlights their role as antimicrobial agents. The phagocytic activity and phagocytic index assays evaluate the ability of immune cells to ingest and eliminate pathogens. The chapter concludes by stressing the importance of standardized methodologies for assessing innate immunity in fish, which are vital for developing effective disease management strategies in aquaculture. These approaches are essential tools for researchers and practitioners in monitoring fish health and improving aquaculture productivity.