Economical and fast analytical methods are required to determine the content of herbs and herbal medicine. With the increasing interest in rose tea and products prepared from rose flower extracts, it has emerged that products prepared from dried rose petals and rose flower extracts should have certain characteristics. In the European Pharmacopeia, there is no specific method to meet the specific needs of the industry. Therefore, in this study, a simple, cheap, fast and reliable RP-HPLC-DAD method was developed and validated for the identification and quantification of flavonoids in the methanol extract of Rosa damascena Mill. (RD) petals. 10 different RD petal samples obtained from Turkey (Isparta), Pakistan and different parts of Iran were studied and compared with fingerprinting. Several trials were conducted to identify the most robust and consistent HPLC method. Separation of flavonoids was achieved on a reversed-phase C-18 column kept at 25 degrees C by gradient elution with a flow rate of 1.2 mL/min, injection volume of 10 mu L and detection at 330 nm. Six different flavonol glycosides were identified which are kaempferol 3-O-glucoside (K3GLU), kaempferol 3-O-galactoside (K3GAL), kaempferol 3-O-rhamnoside (K3RHA), quercetin 3-O-glucoside (Q3GLU), quercetin 3-O-galactoside (Q3GAL), quercetin 3-O-rhamnoside (Q3RHA). K3GLU was determined to be the most suitable compound as a marker and was used for quantification and validation studies. Standard curve of this compound was plotted to calculate quantification. K3GLU amount was found the highest in Isparta rose with 0.7047%. Various validation parameters were done, and the method was found to be specific, stable, linear, sensitive, accurate, precise and robust to determine the chemical specification of RD petals.