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Rose oil (Rowghan-e Gol) is an Iranian traditional medicine used both topical and systemic in gastritis, inflammatory bowel disease, headache, and hemorrhoids. Traditional manuscripts have reported two different methods of preparation for this medicine; the first is macerating rose petals in sesame  oil for  25  days  under  sunlight  (R1),  and  the  second  is  extracting  rose  petals  by squeezing and then boiling the mixture of the extract with sesame oil to evaporate aqueous part (R2). The aim of this article was to study both traditional methods of rose oil preparation in terms of physicochemical factors to evaluate which method is best for industrializing. For this purpose, total phenolics (based on gallic acid), total essential oils (based on citronellol), thin layer chromatography (TLC) profile of the constituents, and oil rancidity indices, i.e., acid and peroxide values were determined through spectrophotometer, gas chromatograph, TLC, and titration, respectively. R1 had greater amounts of total phenolics (0.05% vs. 0.01%). The amount of its essential oil was 15.5 times higher than R2. TLC profiles showed that R1 had one more spots (Rr = 0.04) representing flavonoids (according to natural product indicator). About oil rancidity indices, both samples were in standard ranges but all indices of R1 were greater than R2. It could be due to long exposure of R1 to sunlight. According to the results, R1 had more amounts of flavonoids and essential oils. These compounds are considered as therapeutic agents of rose oil. Therefore, R1 is a more preferable than R2. Appropriate antioxidants should be utilized to protect R1 against sunlight oxidation.