An HPLC-UV method was developed for simultaneously quantifying 13 major components in Semen Oroxyli, a commonly used traditional Chinese medicinal herb, including scutellarin, quercetin-3-O-α-L-arabinopyranoside, oroxin B, chrysin-7-O-β-D-gentiobioside, baicalin, oroxin A, scutellarein, chrysin-7-O-β-D-glucuronide, quercetin, norwogonine, baicalein, chrysin and oroxylin A. The optimal conditions of separation and detection were achieved on an Agilent Zorbax SB-C18 column (250 mm × 4.6 mm, 5 μm) with a gradient elution of acetonitrile and 0.3% (v/v) acetic acid at a flow rate of 1.0 ml/min, with a detection wavelength of 277 nm. A complete separation was obtained within 50 min for the 13 target compounds. All calibration curves showed good linearity (r > 0.9997) within the testing range. The assay was reproducible with overall intra-and inter-day variation of less than 3%. The mean spiked recovery of all analytes was 100 ± 10%, with RSD less than 5%. The validated method was successfully applied to quantitatively analyse 13 flavonoids for quality evaluation of commercial Semen Oroxyli samples from different locations.