Eugenia punicifolia (Kunth) DC, known as “pedra ume-caá” or “pitanguinha do cerrado”, is popularly used to treat infectious diseases like inflammation, and diabetes. Therefore, this study aimed to develop and validate an analytical method by high-performance liquid chromatography with diode array detector (HPLC-DAD) for the seasonal quantification of gallic acid (GA) and ellagic acid (EA), and to co-validate the method for quantification from the ethanol extract and fractions of the E. punicifolia leaves. Chromatographic separations were performed with a mobile phase gradient of acetonitrile/methanol/water acidified with 0.2% formic acid, a flow of 0.8 mL min 1, detection at 254 nm, and a C18 column (250 × 4.6 mm, 5 µm). Method validation parameters were determined following Brazilian legislation. The HPLC-DAD analytical method developed for the quantification of GA and EA was validated and demonstrated to be selective, linear, precise, accurate, robust, and without matrix effect. Accordingly, it is helpful for the analysis of these tannins in crude ethanol extracts and fractions from E. punicifoila leaves. Thus, our results may be of great help for quality control assessment and standardization of raw materials containing Eugenia punicifolia.