Purpose: This study established a high-performance liquid chromatography (HPLC) method to standardize pentagalloylglucose as a marker compound in <i>Paeonia lactiflora</i> (<i>P. lactiflora</i> ) root extracts. Methods: The analytical procedure for standardizing pentagalloylglucose was optimized using HPLC on a C18 column with a gradient elution of 0.1% trifluoroacetic acid and acetonitrile. The method was validated for specificity, linearity, limit of detection (LOD), limit of quantitation (LOQ), accuracy, and precision. Results: The method demonstrated high linearity, as evident from a correlation coefficient R<sup>2</sup> of 0.9999. The LOD and LOQ were 0.92 and 2.78 μg/mL, respectively. Recovery was determined to be in the range of 101.15 ± 0.49–102.30±0.13, with the relative standard deviation (RSD) values between 0.12%–0.50%, indicating high accuracy. Conclusion: The research lays a foundation for the production of functional foods or cosmetics using pentagalloylglucose derived from <i>P. lactiflora</i> root extract.