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Jitong Ning Tablet (JTNT), a traditional Chinese herbal formula, consists of Eucommia ulmodies oliv, Angelicae pubescentis radix, Aconiti radix cocta, Corydalis yanhusuo w.t. wang, Glycyrrhizae radix et rhizoma, Paeoniae radix rubra and Radix puerariae. It has been demonstrated to show protective effects on ankylosing spondylitis and anti-inflammatory effects. The chemical compositions of JTNT, playing a key role in quality control, remain unknown. In this study, an ultra-performance liquid chromatography combined with quadrupole time of flight mass spectrometry (UPLC-Q–TOF–MS) method in both positive and negative ion mode was established to investigate the chemical constituents of JTNT formula. In total, 162 compounds including flavonoids, triterpenoids, coumarins, alkaloids, phenylpropionic acids, lignans, terpenoids, and organic acids were detected, 152 of which were unambiguously or tentatively identified by comparing their retention times and accurate mass measurement with reference compounds and data in literatures. Our results would benefit quality control and chemical basis for JTNT.Development of an UPLC-Q–TOF–MS for chemical profile of Jitong Ning Tablet.A total of 162 compounds were identified in Jitong Ning Tablet.The fragmentation pathways of major types of compounds were characterized.