A new method was developed for the simultaneous determination of nine major constituents in Dracocephalum rupestre, including 5,7-dihydroxychromone (1), eriodictyol-7-O-β-D-glucoside (2), luteolin-7-O-β-D-glucoside (3), naringenin-7-O-β-D-glucoside (4), apigenin-7-O-β-D-glucoside (5), eriodictyol (6), luteolin (7), naringenin (8) and apigenin (9). The quantitative determination was conducted by reversed phase high-performance liquid chromatography with photodiode array detector (LC–PDA). Separation was performed on an Agilent Eclipse XDB-C18 column (150 mm × 4.6 mm i.d., 5 μm) with gradient elution of acetonitrile and 0.5% aqueous acetic acid. The components were identified by retention time, ultraviolet (UV) spectra and quantified by LC–PDA at 260 nm. All calibration curves showed good linearity (r2 > 0.999) within test ranges. The reproducibility was evaluated by intra- and inter-day assays and R.S.D. values were less than 3.0%. The recoveries were between 95.15 and 104.45%. The limits of detection (LOD) ranged from 0.002 to 0.422 μg/ml and limits of quantification (LOQ) ranged from 0.005 to 1.208 μg/ml, respectively. The identity of the peaks was further confirmed by high-performance liquid chromatography with triple-quadrupole mass spectrometry system coupled with electrospray ionization (ESI) interface. The developed method was applied to the determination of nine constituents in 14 samples of D. rupestre collected at various harvesting times. Most compounds accumulated at much higher amounts in about June–July. The satisfactory results indicated that the developed method was readily utilized as a quality control method for D. rupestre.