<![CDATA[Vaccinium bracteatum Thunb. (VB) is a species of Vaccinium with a high flavonoid content and broad pharmacological potential, including antioxidant, anti-inflammatory, and neuroprotective activity. This study aims to isolate and characterize the main bioactive compounds from VB leaf water extract, as well as to develop and validate analytical methods for quantification of marker compounds as the basis for standardization of extracts. VB leaves are collected from various locations in South Korea and extracted using the aqueous extraction method. The compounds were then separated through silica gel column chromatography and HPLC preparations, and identified using UV-Vis, FTIR, 1D/2D NMR, and LC-MS/MS. HPLC-PDA AND LC-MS/MS methods were validated according to ICH guidelines (linearity, accuracy, precision, LOD, LOQ, specificity). The isolation results showed five main compounds: the flavonoids orientin (2.35%) and isoorientin (1.87%), phenolic acid vanillic acid (0.95%) and protocatechuic acid (1.12%), as well as the glycoside (0.78%). Validation of the analytical method showed a linearity of R² > 0.999, an accuracy of 98.9–101.2%, a precision of <2%, and a low LOD/LOQ, supporting the sensitive quantification of major and minor compounds. Spectroscopic and LC-MS/MS characterizations confirm the chemical structure of the compounds, confirming the purity of the isolate. These findings provide a scientific basis for the standardization of VB leaf extracts, supporting the development of herbal or nutraceutical products based on flavonoids and glycosides. Further research is recommended to explore in vivo activity, minor metabolite interactions, as well as the relationship of biosynthetic gene expression with phytochemical profiles to maximize the therapeutic potential of VB.]]>
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