The use of antibiotics in food-producing animals has generated considerable interest because the widespread administration of these drugs may lead to the development of resistant human pathogens. A large increase in the demand for seafood products has occurred in the last century. This has led to a concomitant increase in high-intensity aquaculture methods, characterized by high stock density and volume, and the heavy use of formulated feeds containing antibiotics, among other substances. Therefore, accurate and sensitive determination of antibiotic residues is now a necessity. In order to protect human health, the European Union and other regulatory authorities worldwide have established maximum residue limits (MRL) for antibiotic residues in animal products entering the human food chain. This paper reviews the most recent methods for analysis of antibiotic residues in fish.