A　simple,　sensitive　and　reproducible　ultra-performance　liquid　chromatography-tandem　mass　spectrometry　method　has　been　developed　for　the　simultaneous　determination　of　atenolol,　a　β-adrenergic　receptor-blocker　and　chlorthalidone,　a　monosulfonamyl　diuretic　in　human　plasma,　using　atenolol-d7　and　chlorthalidone-d4　as　the　internal　standards　(ISs).　Following　solid-phase　extraction　on　Phenomenex　Strata-X　cartridges　using　100μL　human　plasma　sample,　the　analytes　and　ISs　were　separated　on　an　Acquity　UPLC　BEH　C18　(50mm×2.1mm,　1.7μm)　column　using　a　mobile　phase　consisting　of　0.1%　formic　acid-acetonitrile　(25:75,　v/v).　A　tandem　mass　spectrometer　equipped　with　electrospray　ionization　was　used　as　a　detector　in　the　positive　ionization　mode　for　both　analytes.　The　linear　concentration　range　was　established　as　0.50-500ng/mL　for　atenolol　and　0.25-150ng/mL　for　chlorthalidone.　Extraction　recoveries　were　within　95-103%　and　ion　suppression/enhancement,　expressed　as　IS-normalized　matrix　factors,　ranged　from　0.95　to　1.06　for　both　the　analytes.　Intra-batch　and　inter-batch　precision　(CV)　and　accuracy　values　were　2.37-5.91　and　96.1-103.2%,　respectively.　Stability　of　analytes　in　plasma　was　evaluated　under　different　conditions,　such　as　bench-top,　freeze-thaw,　dry　and　wet　extract　and　long-term.　The　developed　method　was　superior　to　the　existing　methods　for　the　simultaneous　determination　of　atenolol　and　chlorthalidone　in　human　plasma　with　respect　to　the　sensitivity,　chromatographic　analysis　time　and　plasma　volume　for　processing.　Further,　it　was　successfully　applied　to　support　a　bioequivalence　study　of　50mg　atenolol+12.5mg　chlorthalidone　in　28　healthy　Indian　subjects.　©　2016　John　Wiley　&　Sons,　Ltd.