In chemical studies or quality control of extracts from medicinal plants and herbal products, high performance liquid chromatography (HPLC) constitutes the most used technique for separation, isolation, quantification and identification of secondary metabolites. Most of these methods of analysis by HPLC use toxic solvents such as acetonitrile and methanol and, therefore, the amount of toxic waste generated is high, requiring expensive treatments to minimize damage to the environment. In addition, the manipulation of these solvents may result in serious harm to the health of the analyst. Based on Green Chemistry concepts, which aims to decrease and/or replace these toxic solvents by non-toxic ones, it is possible to develop environmentally friendly ("green") methods of analysis. In this context, this study aims to determine chromatographic parameters with the greatest effects on the separation of the secondary metabolites present in the hydroalcoholic extract of Byrsonima intermedia leaves (used in folk medicine by its antiseptic, antimicrobial, anti-hemorrhagic, anti-inflammatory, analgesic, diuretic, antiasthmatic and cicatrizing properties) by HPLC using ethanol and water as mobile phase, to assist in the development of an environmentally friendly method. Fractional factorial design was carried out in the screening of variables [initial ethanol % (X1); final ethanol % (X2); temperature of analysis (X3); acid % in water (X4) and flow-rate (X5)], using the number of peaks and their distribution in the chromatogram, applying the GCFR (Green Chromatographic Fingerprinting Response) response function. The hydroalcoholic extract from the leaves of B. intermedia was submitted to clean-up with Sep-Pack C18 (Strata Phenomenex) and 20 μL were injected into HPLC system [Jasco chromatograph; Phenomenex Luna C18 column (250 x 4.6 mm, 5 μm); photodiode array detector (PAD)] in the resultant conditions from the experimental design plus 4 replicates at central point, totalizing 20 analysis. After treatment of the obtained data it was identified that the factors (X2), (X3) and (X5) influenced in 71% in the distribution of the peaks. These results will aid in the development of a green and sustainable method for analyzing the hydroalcoholic extract of B. intermedia by HPLC using water and ethanol as the mobile phase.