Abstract Background Aquaculture is one of the worldwide strategic development fields, and its importance is evident in its significant worldwide growth in the last decades. This growth is associated with the implementation of intensive and semi-intensive production methods, with the use of antibiotics in order to prevent the emergence and spread of infectious diseases in fish. Fluoroquinolones, tetracyclines, and sulfonamides, among others, are widely used for this purpose. This practice constitutes a real public health concern, not only due to the presence of antimicrobial residues in edible tissues, which can cause allergic reactions in hypersensitive individuals, but also due to the emergence of bacterial resistance. Consequently, the European Union's Regulatory Agencies have established maximum residue limits and specific requirements regarding the performance of analytical methods. Scope and approach This article reviews the most recent analytical methodologies concerning antimicrobial residues in fish, reported in the literature, given emphasis on sample procedures, extraction/purification methods, chromatographic conditions and validation techniques according to legislation. Key findings and conclusions Liquid chromatography coupled to mass spectrometric detection is used as preferential tool in the analysis of antimicrobial residues in fish. The current analytical strategy is shifting towards multi-residue and multiclass methods, which save time, and surely represent the future trend in this field. The extraction process still represents the limiting factor of any multi-residual method, since it should provide acceptable recovery of all analytes with a broad range of physicochemical properties, and therefore this is probably the step that requires more in-depth research.