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We developed and validated a liquid chromatography\u2013tandem mass spectrometry method for the simultaneous determination of 54 analytes in human serum, including antiepileptic drugs, antidepressants, antipsychotics, benzodiazepines, antifungal agents, theophylline, and selected clinically relevant metabolites. Serum samples were prepared by protein precipitation with perchloric acid. Chromatographic separation was achieved on an ACQUITY UPLC BEH C18 column using water\/methanol mobile phases containing 2\u00a0mM ammonium acetate. Detection was performed in polarity\u2010switching selected reaction monitoring mode using two transitions per analyte and 28 isotope\u2010labeled or structurally related internal standards. The total run time was 8.5\u00a0min. Analyte\u2010specific calibration models and validated concentration ranges covered 0.1\u201322,500\u00a0ng\/mL, depending on the analyte. The method demonstrated acceptable selectivity, calibration performance, accuracy, precision, matrix effects, and stability under routine handling and storage conditions for analytes with available data. Within\u2010run and between\u2010run precision did not exceed 14.3% and 13.9%, respectively. Matrix effects ranged from \u22128.35% to +10.25%. Application to 120 remnant patient serum samples supported the feasibility of multiplex measurement of drugs from several therapeutic classes in a single analytical run. The method is suitable for routine serum\u2010based therapeutic drug monitoring and for targeted toxicological interpretation of a predefined analyte panel.<\/jats:p>","DOI":"10.1002\/jssc.70560","type":"journal-article","created":{"date-parts":[[2026,10,21]],"date-time":"2026-10-21T15:04:09Z","timestamp":1792595049000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["A Validated Multi\u2010Analyte Liquid Chromatography\u2013Tandem Mass Spectrometry Method for Simultaneous Therapeutic Drug Monitoring and Targeted Toxicological Assessment of 54 Drugs and Metabolites in Human Serum"],"prefix":"10.1002","volume":"49","author":[{"given":"Katerina","family":"Kratka","sequence":"first","affiliation":[{"name":"Institute of Laboratory Medicine Faculty of Medicine University of Ostrava  Ostrava Czech Republic"},{"name":"Institute of Laboratory Medicine University Hospital Ostrava  Ostrava Czech Republic"},{"name":"Lab4Life 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