Berries are a rich source of natural antioxidant compounds, which are essential to profile, as they add to their nutritional value. However, the complexity of the matrix and the structural diversity of these compounds pose challenges in extraction and chromatographic separation. By relying on multivariate curve resolution alternating least squares (MCR-ALS) ability to extract components from complex spectral mixtures, our study evaluates the contributions of various extraction techniques to interference, extractability, and quantifying different groups of overlapping compounds using liquid chromatography diode array detection (LC-DAD) data. Additionally, the combination of these methods extends its applicability to evaluate polyphenol degradation in stored berry smoothies, where evolving factor analysis (EFA) is also used to elucidate degradation products. Results indicate that among the extraction techniques, ultrasonication-assisted extraction employing 1% formic acid in methanol demonstrated superior extractability and selectivity for the different phenolic compound groups, compared with both pressurized liquid extraction and centrifugation of the fresh berry smoothie. Employing MCR-ALS on the LC-DAD data enabled reliable estimation of total amounts of compound classes with high spectral overlaps. Degradation studies revealed significant temperature-dependent effects on anthocyanins, with at least 50% degradation after 7 months of storage at room temperature, while refrigeration and freezing maintained fair stability for at least 12 months. The EFA model estimated phenolic derivatives as the main possible degradation products. These findings enhance the reliability of quantifying polyphenolic compounds and understanding their stability during the storage of berry products.Graphical abstract