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The National Formulary, USP 34 NF 29, (2011) 23 Leilei G., Xiangqin T., Shangran S., Jian H., Xiaojun S., Suying M.; Simultaneous determination of florphenicol and diclazuril in compound powder by RP-HPLC-UV method; Journal of Chemistry, (2014); 2014: 1–5. 24 ICH Harmonised Tripartite Guideline, Validation of Analytical Procedures: Text and Methodology Q2(R1). Geneva, (2005). http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Quality/Q2_R1/Step4/Q2_R1__Guideline.pdf.https://doi.org/10.1093/chromsci/bmv095https://t.ly/BJVjpMSA Google ScholarTwo chromatographic methods were developed, optimized and validated for the simultaneous determination of three approved aquaculture antimicrobials, namely sulphadimethoxine sodium, trimethoprim and florphenicol in fish tissues. The developed methods were based on simple liquid extraction technique. The first method employs thin-layer chromatography as a clean-up procedure coupled with densitometric determination for the separated drugs. The second method is an HPLC one using X-Terra™ C18 column. Several mobile-phase systems and extracting solvents were tried to optimize the separation and the extraction procedures from fish tissues. The procedures were applied for the analysis of spiked fish tissue samples at three different concentration levels (10, 50 and 100 ppm). A comparative study was conducted between the proposed methods to discuss the advantage of each one. The methods were validated according to the international conference on harmonization guidelines. The proposed methods were successfully applied for the determination of the studied drugs in spiked fish tissues, pure powders and in their veterinary pharmaceutical formulation.enUniversity of Chromatographic TechniquesApproved AntimicrobialsArticlehttps://doi.org/10.1093/chromsci/bmv095