Recently, it has been demonstrated that a rare flavonoid, 5,7-dihydroxy-6,8,4′-trimethoxyflavonol (1), isolated from the leaves of Rubus rosifolius showed significant antiproliferative activity against different cancer cell lines and had a diuretic effect. This study aimed to determine the best extraction method evaluating dynamic maceration extraction (DME), ultrasonic assisted extraction (UAE), and microwave assisted extraction (MAE). In addition, level optimization for different factors was focused using Design of Experiments. The flavonoid content was estimated by high performance liquid chromatography (HPLC). The results indicated that UAE provided the highest content with the shortest extraction time. For optimization, response surface methodology (RSM) was used, applying a Box-Behnken Design (BBD) with three factors at three levels. It was possible to observe that the flavonoid content significantly increased with the increase of temperature (max. 70°C). The time had little influence on the extraction and, for this reason, was set at 25 min. With regard to the drug/solvent ratio, it was observed that this had an impact on yield with a ratio of 3.5%. A model was built and a determination coefficient of 0.98 was obtained. After validation, an optimized method was developed, able to extract 1 with a lower time, lower solvent consumption, and affording higher contents.