Punica granatum, "pomegranate", belongs to the botanical family Punicaceae. Among the constituents present in P. granatum peel are tannins, including punicalina, punicalagina, among others [1]. Pomegranate peel extract is rich in polyphenols, which have shown a strong antiseptic effect and antibacterial activity [2]. The most widely used technique for separating and identifying bioactive compounds of P. granatum is the high-performance liquid chromatography (HPLC). Despite the efficiency of HPLC, most studies use toxic organic solvents such as acetonitrile and methanol that generate large amounts of waste requiring high cost treatments and pose risks to the health of the analyst. Thus, green and sustainable solvents such as ethanol enable the substitution of these toxic organic solvents. Therefore, the objective of this work was to determine the important chromatographic parameters for obtaining the chromatographic fingerprinting from the peel of P. granatum fruit utilizing green chemistry concepts. The dried peel of P. granatum (pomegranate) were obtained commercially from the manufacturer “Essencia do ser Produtos Naturais Ltda” (lot: A013808). The infusion was prepared with 1.0 g of powder dried pomegranate peel and 10 mL of water at 80 ° C. After extraction, the solution was filtered and applied in PTFE microfilter discs of 0.45 μm (Phenomenex®). Aliquots of 20 μL were analyzed by HPLC coupled to Photodiode Array Detector (HPLC-PAD) (Jasco®, PU pump 2089 plus, PAD MD 2010 plus detector, injector automatic AS 2055 plus) with Waters® XBridge RP column (150 x 4.6 mm x 4 μm) and pre-column (4x3 mm, 5μm). The variables were sorted using Fractional Factorial Design of 2 levels and 5 factors (2v5-1). Two responses were evaluated: (1) the total number peak in the chromatogram and (2) the distribution of the same time along of the chromatogram (Green Chromatographic Factor Response (GCFR) [3]. The factors and levels investigated in the screening were: initial % of EtOH (X1), final % of EtOH (X2), temperature (°C) of the analyzes (X3), acetic acid % in water (X4) and flow-rate (mL/min) of the mobile phase (X5). The statistical analyzes were performed on Octave® software. The significant effects were those of factors X1, X3 and X5. Therefore, these factors are those that significantly interfere in the separation of the substances present in the infusion of P. granatum, which can help in the development of a green and sustainable method to obtain the P. granatum fingerprinting by HPLC, using water and EtOH as mobile phase.