An HPLC technique with UV spectrophotometric detection was developed for qualitative and quantitative determination of vitexin, rutin, hyperoside, and quercetin in Crataegus sanguinea hawthorn herbs (flowers and leaves). The separation used a Luna C18(2) column (4.6 × 150 mm, 5 μm) and a stepwise gradient. Calibration curves for the four flavonoids exhibited good linearity (r2> 0.995) in the studied ranges. The analytical recoveries over one day/week were 95.9-104.3%/95.6 – 102.9% for vitexin; 94.0 – 102.2%/93.5 – 101.5% for rutin; 98.0 – 102.2%/97.5 – 102.9% for hyperoside; and 96.0 – 109.8%/97.2 – 111.0% for quercetin. The intra- and interday coefficients of variation (RSD, %) were <3%. The relative error (ε, %) of a single determination at confidence level 95% was <3% for rutin, hyperoside, and vitexin and <4% for quercetin. © 2017, Springer Science+Business Media, LLC.