IntroductionSystematic analyses of naphthoquinones in Juglans cathayensis have not yet been reported. It is very challenging to identify naphthoquinones with various structural diversities, especially those at trace levels. ObjectiveTo develop an efficient analytical approach for systematic discovery and identification of naphthoquinones in Juglans cathayensis. MethodologyA novel four-step approach was evaluated by utilizing various scan functions of liquid chromatography-triple quadrupole-linear ion trap mass spectrometry (LC-QTRAP-MS/MS) and liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (LC-QTOF-MS/MS) along with data mining strategies. First, MS/MS fragmentation behaviors of naphthoquinones were investigated. Second, multiple ion monitoring triggered enhanced product ion scan (MIM-EPI) with specified ions was conducted to identify targeted naphthoquinones. Third, other scan functions of QTRAP-MS/MS and data mining strategies were explored to identify untargeted naphthoquinones. Fourth, structural rationalization and confirmation of naphthoquinones were performed using QTOF-MS/MS via its accurate mass measurement and MS/MS fragmentation functions. ResultsOptimal scan methods and data mining strategies using QTRAP-MS/MS were obtained for identification of targeted and untargeted naphthoquinones. Consequently, 48 naphthoquinones including 24 novel ones were identified or tentatively identified from Juglans cathayensis. ConclusionA novel four-step approach for efficient discovery and identification of naphthoquinones was developed by exploring various scan functions of current LC-MS/MS technologies and data mining strategies, providing an example for systematic characterization of certain classes of phytochemicals, especially trace analytes in complex samples. Copyright (c) 2015 John Wiley & Sons, Ltd. In order to systematically identify naphthoquinones in J. cathayensis, a novel four-step approach for efficient discovery and identification of naphthoquinones was developed by exploring various scan functions of current LC-MS/MS technologies and data mining strategies. Consequently, 48 naphthoquinones including 24 novel ones were identified or tentatively identified from J. cathayensis. The present study provided an example for systematic characterization of certain classes of phytochemicals, especially trace analytes in complex samples.