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Background Anesthetics and sedatives are frequently used to prevent abrasions caused by stress and facilitate fish management. However, drug residues may persist and cause changes in fish conditions and induce side effects. In addition, drugs that are not permitted for use in edible fish are sometimes potentially used in fish. The drugs can also be found in wastewater and are likely to be detected in fish. Objective The purpose of this study was to establish a quantitative analytical method for 10 anesthetic and sedative (azaperone, chlorpromazine, diazepam, estazolam, haloperidol, nitrazepam, nordiazepam, oxazepam, perphenazine, and temazepam) residues in fish sold in Korean markets. Methods Shrimp, flounder, and eel samples were selected as matrices. Acetonitrile (ACN) containing 0.1% formic acid was selected as an extraction solvent for shrimp and 100% ACN for flounder and eel. The QuEChERS method with C18 and PSA was used as the extraction procedure, and the analysis was performed by LC-MS/MS. Results Limit of quantitation, recovery, accuracy, and precision were validated, and satisfactory results were obtained for the drugs. All results applied to the real samples were negative. Conclusion An optimal validation method was studied. Since the results for all samples were negative, it is considered that additional studies are needed by increasing the number of drugs. Highlights The most effective QuEChERS pretreatment method and conditions of LC-MS/MS for the analysis of anesthetics and sedatives in fish were established.