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This research focuses on developing and validating a UPLC\u2010MS\/MS method for the simultaneous determination of bisoprolol and hydrochlorothiazide in human plasma, adhering to established regulatory guidelines for bioanalytical method validation. The development and validation focus on creating a robust and sensitive assay suitable for bioequivalence studies and routine therapeutic drug monitoring. This method utilizes bisoprolol D5 and hydrochlorothiazide C13 D2 as internal standards to enhance accuracy and precision. Chromatographic separation was achieved on a Waters Acquity UPLC BEH C18 column (100\u2009\u00d7\u20092.1\u2009mm, 1.7\u2009\u03bcm particle size) with an isocratic mobile phase consisting of 10\u2009mM ammonium formate buffer, methanol, and 0.1% ammonia solution (10:90, v\/v). The flow rate was set at 0.3\u2009mL\/min, with a retention time of 2.2\u2009min. Multiple reaction monitoring (MRM) was used with positive ESI for bisoprolol (m\/z 326.36\u2009\u2192\u2009116.13, internal standard m\/z 331.36\u2009\u2192\u2009121.13) and negative ESI for hydrochlorothiazide (m\/z 296.11\u2009\u2192\u2009269.00, internal standard m\/z 299.11\u2009\u2192\u2009270.00). The analytes and their respective internal standards were co\u2010extracted using a liquid\u2013liquid extraction method with <jats:italic>tert<\/jats:italic>\u2010butyl methyl ether as the extraction solvent. Linearity for bisoprolol and hydrochlorothiazide was maintained over a concentration range of 1\u2013100\u2009ng\/mL for bisoprolol and 1.0\u2013300\u2009ng\/mL for hydrochlorothiazide, respectively, using a weighted least squares linear regression model (1\/x). This method achieved a lower limit of quantification (LLOQ) of 1.0\u2009ng\/mL, making it highly sensitive for the detection of these analytes. Moreover, the method demonstrated high accuracy, precision, selectivity, and reduced overall analysis time, making it well suited for routine analysis and bioequivalence studies of 10\u2009mg bisoprolol and 25\u2009mg hydrochlorothiazide tablets.<\/jats:p>","DOI":"10.1002\/jms.5159","type":"journal-article","created":{"date-parts":[[2025,7,28]],"date-time":"2025-07-28T04:19:53Z","timestamp":1753676393000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Development and Validation of an UPLC\u2010MS\/MS Method for Simultaneous Quantification of Bisoprolol and Hydrochlorothiazide in Human Plasma"],"prefix":"10.1002","volume":"60","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-0751-6301","authenticated-orcid":false,"given":"Asmaa","family":"Al\u2010Asmar","sequence":"first","affiliation":[{"name":"Faculty of Science An\u2010Najah National University  Nablus Palestine"},{"name":"Energy, Water and Food Security 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