Folium Turpiniae has been used as a traditional Chinese medicine for the treatment of abscesses, fevers, gastric ulcers, and inflammations. This paper describes a strategy of combining HPLC with photodiode array detection and quadrupole TOF-MS, as well as phytochemical and chemometrics analysis for the characterization, isolation, and simultaneous quantification of the chemical constituents of Folium Turpiniae. 19 constituents were identified, namely, 11 flavonoids, seven gallic acid derivates, and quinic acid. Among them, 15 compounds were identified in this herbal medicine for the first time; compound 10 appears to be novel and was isolated and confirmed as ellagic acid-3-O-α-L-rhamnopyranoside by NMR spectroscopy and MS. In addition, nine marker compounds, namely gallic acid (2), ellagic acid-3-O-α-L-rhamnopyranoside (10), apigenin-7-O-(2''-rhamnosyl)gentiobioside (11), ellagic acid (12), luteolin-7-O-β-D-neohesperidoside (13), ligustroflavone (14), 4'-O-methylellagic acid-3-O-α-L-rhamnopyranoside (16), rhoifolin (17), and neobudofficide (18), were quantified simultaneously in ten batches of Folium Turpiniae collected from different regions. Moreover, hierarchical clustering analysis and principal component analysis indicated that both samples from Hubei (S1) and Guangxi (S3) provinces showed apparent differences from the others. Samples from Jiangxi province (S2, S4, and S7-10) possessed similar properties and therefore belong to the same group.