Lophatheri Herba (Danzhuye; LH), the dried leaves of Lophatherum gracile Brongn (Poaceae), is commonly used in Chinese herbal medicine as an antipyretic, antibacterial, and diuretic. Chemical analysis has been conducted to isolate and identify seven major flavonoid glycosides, including a new flavone C-glycoside, luteolin 6-C-β-D-glucuronopyranosyl-(1→2)-β-D-glucopyranoside (1), isoorientin (2), swertiajaponin (3), luteolin 6-C-β-D-glucuronopyranosyl-(1→2)-α-L-arabinopyranoside (4), isovitexin (5), swertisin (6), luteolin 7-O-β-D-glucopyranoside (7), and luteolin 6-C-α-L-arabinopyranoside (8), from commercially available LHs in Taiwan. The structure of the new compound (1), the maximum component, was determined by extensive one- (1D-) and two-dimensional (2D-) nuclear magnetic resonance (NMR), and MS spectral analyses. The 1H and 13C-NMR of two rotameric pairs of 3 and 6 were also assigned. To establish the quality control platform of LH, we developed a simultaneous determination of multiple components in 10 commercially available LHs, collected from different areas of Taiwan, by high performance liquid chromatography (HPLC) and ultra performance liquid chromatography (UPLC), as well as quantitative measurement of the major components 1–4, and 8. All isolated major compounds showed good linear regression (R2 ≥ 0.9993) within the test ranges and high reproducibility. These methods are readily accessible for the quality control of LH.