The objective of this study was to create a liquid chromatographic method that could accurately and simultaneously quantify the amount of Quercetin, Berberine, and Phytosterol present in lyophilized liposome used to treat metabolic syndrome. To achieve this goal, the team utilized a Shim‐pack GIST C8 column with a gradient elution of 0.1% (V/V) triethylamine buffer: acetonitrile at 30°C. They analyzed the phytoconstituents using an eluent flow rate of 1 mL/min and ultraviolet detection at 205 nm. The method was linear within a range of 25–77 μg/mL, which had excellent correlation coefficients (R2) of 0.9998, 0.9998, and 0.9996 for Quercetin, Berberine, and Phytosterol, respectively. The %RSD for precision and robustness of the method were also less than 2%, and the recovery values for Quercetin, Berberine, and Phytosterol were 99.5%, 100.5%, and 99.8%, respectively. The assay values of were found to be 100.2%, 101.6%, and 100.5% for Quercetin, Berberine, and Phytosterol, loaded lyophilized liposomes, respectively. As a result, this specific, accurate, and precise method can be employed for the routine analysis of Quercetin, Berberine, and Phytosterols in QC laboratories.