Objectives: The objective of this work was to investigate the effect of reversed-phase high-performance liquid chromatography conditions for the determination of flavonoids in the plant extract. Methods: The analytical conditions (total flow rate, mobile phase, colum temperature, and detection wavelength) optimized for gallic acid, rutin, and quercetin. The optimized method was compared with the control method and evaluated with the use of reference compounds. Results: The result shows that the maximum flavonoids recovery was in observed on modified chromatographic conditions, Colum: Octadecyl-silica C18column (250 mm × 4 mm, 5 μm) Mobile Phase solution water-acetic acid (25:1) and methanol. Mobile Phase solution B was a Mobile phase – 4th min - 20%, 10th min - 80%, and a flow rate of 0.5 mL/min. Colum temperature 35°C and detection wavelength was 260 nm. Conclusion: In this work, the proposed method will increase the analysis time but minimize the mobile solvents and have the potential of being useful for the routine analysis of flavonoids.