Phytochemicals are chemical compounds found in some plants that have medical significance and have a specific physiological effect on humans. In order to obtain pure compounds, it is common practice to use a variety of separation techniques, including Thin-Layer Chromatography (TLC) and High-Performance Liquid Chromatography (HPLC). The TLC analysis of Paeonia officinalis was performed for estimation of phenol & flavonoid. In case of flavonoid quercetin was used as standard. The Rf value for quercetin was found to be 0.60. The ethanolic extract possessed quercetin as flavonoid which is evident by Rf value. Further the aqueous extract can be supposed to have flavonoid other than quercetin. The results of TLC for phenol point towards fact that ethanolic extract contain gallic acid as phenol which is in accordance with the Rf value. Further, calibration curve of Quercetin was plotted by using HPLC. The sample was then passed in HPLC to analyze the flavonoids in sample. It was observed that for quercetin standard the Retention time was found to be 3.017. While in case of Paeonia officinalis extract retention time was noted to be 2.953, with the % assay of 0.27%. The study showed that the Paeonia officinalis are potential sources of phytochemicals that promote health and have strong biological activity that are therapeutically relevant.