This study outlines the development of an innovative method utilizing UHPLC-PDA for the simultaneous identification of polyphenols and bitter acids (alpha-acids, beta-acids, and iso-alpha-acids) in beer samples. The resulting chemical profiles were leveraged to distinguish the characteristics of four bergamot-flavored beers, each representing distinct styles (IPA, Lager, Blanche, and ALE), produced on a pilot-scale plant. In a streamlined 29-minute analysis, twenty-nine polyphenols and fourteen bitter acids were successfully identified under optimized separation conditions. Rigorous validation, encompassing parameters such as Limit of Detection (LOD), Limit of Quantification (LOQ), R-squared (R2), repeatability, and reproducibility, was conducted for polyphenol quantification using constructed calibration curves with seven levels. Exploring polyphenolic components as potential discriminators among different beer styles, a total of thirty-two polyphenolic compounds were identified and quantified, including characteristic bergamot peel polyphenols like neoeriocitrin, naringin, and neohesperidin. The determination of bitter units and total phenols further aided in style differentiation. Multivariate analysis provided additional insights into variations among specific beer styles, revealing discrepancies in the presence or relative concentrations of specific compounds linked to brewing ingredients and processes. This research enhances to trace the fingerprinting of the chemistry governing beer quality through a straightforward and cost-effective analytical approach, readily applicable in common laboratories.