Abstract <p>The principal aim of this investigation is to improve the precision and accuracy of quantifying sugars, such as glucose, sucrose, and fructose, through the utilization of hydrophilic interaction liquid chromatography (<b>HILIC</b>) with charged aerosol detection (<b>CAD</b>) in <i>Pueraria tuberosa</i>, a plant renowned for its pharmacological properties. Refractive index detectors are commonly employed to determine sugars in plant extracts. However, it is important to note that these detectors suffer from poor sensitivity and are prone to interference from co-eluting components. Therefore, alternative methods need to be explored to overcome these limitations. HPLC-CAD offers a promising solution by providing improved sensitivity, enhanced precision, and reduced interference from co-eluting sample components. Optimal separation of substances was achieved by implementing an Inertsil HILIC column (250 × 4.6 mm, 5 μm). The mobile phase consisted of 0.1% trifluoroacetic acid in acetonitrile and 0.1% trifluoroacetic acid in water. The flow rate was set at 0.8 mL/min. By employing a systematic approach to modify the chromatographic conditions, this study has successfully improved the sensitivity, resolution, and reliability of sugar determination.</p>