Liquid chromatography-electrospray ionization quadrupole time-of-flight tandem mass spectrometry (LC-ESI-QTOF-MS) was applied to identify chemical components occurring in Neopicrorhiza scrphulariiflora roots. 24 compounds, including 12 iridoid glycosides, 3 phenyl glycosides and 9 phenylethanoid glycosides, were detected and tentatively characterized according to accurate mass measurements and their characteristic fragmentation at low and high collision energy. Meanwhile, appropriate fragmentation pathways were proposed based on definite composition of the product ions. 6′-O-Feruloyl-3,4-dihydrocatalpol and 1-[2-(4-(3,4-dihydroxycinnamoyl)-3-glucosyl)glucosy]ethy-3-hydroxy-4-methoxybenzene are potentially novel compounds. 6′-O-Caffeoylcatalpol and maxoside were characterized in N. scrphulariiflora for the first time. This study established that the LC-ESI-QTOF-MS method is efficient for identifying, and could be the basis for the comprehensive quality control of, N. scrphulariiflora roots.