Soybean, Glycine max, belonging to the Fabaceae family is one of the most important crops in the world, not only because it is an important source of oil and protein, but also of secondary metabolites [1]. Among its secondary metabolites in particular are isoflavones, such as genistein and daidzein, which play an important role in the prevention of chronic diseases, presenting an anticancer effect and antioxidant activity. The most widely used technique for separating and identifying bioactive compounds of G. max is the high-performance liquid chromatography (HPLC). However, most papers use toxic organic solvents, such as acetonitrile and methanol, as HPLC mobile phase that generate large amounts of waste and can provide risks to the health of the analyst and the environment. In this context, the development of green and sustainable chromatographic methodologies for the study of herbal or phytotherapeutic plants, with the use of reagents environmentally friendly would be a desirable strategy [2]. Therefore, the objective of this work was to determine the important chromatographic parameters for obtaining the chromatographic fingerprinting from the seeds of G. max utilizing green chemistry concepts. Soybean were obtained commercially from the manufacturer “MãeTerra Produtos Naturais Ltda”. The extract was prepared using 50 g of the ground powder in 500 mL of EtOH/H2O (7:3, v/v) for 144 h, exchanging the solvent every 48 h and the organic solvent was evaporated to dryness in rotatory evaporator to afford the crude extract (8.40g). Then, 50 mg of the solubilized extract in 2 mL EtOH/H2O (7:3) were applied to a C18 cartridge (Phenomenex®, Strata, 500 mg-3mL). The collected fraction was then filtered on PTFE membrane disc (Allcrom®, 25 mm) with 0.45 μm pores and 20 mL injected into the Jasco® brand HPLC couple to Photodiode Array Detector (HPLC-PAD) system with a XBridge reverse phase column (150 x 4.6 mm) and guard-column (4x3 mm, 5 μm). Variable screening was performed using 2-level and 5-factor Fractional Factorial Design (2v5-1) and calculations made in the Octave® software. Two responses were evaluated: (1) the total number of peaks in the chromatogram and (2) the distribution of the same along the 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 analyzes (X3), acetic acid % in solvent A (X4) and flow-rate (mL/min) of the mobile phase (X5). The significant effects in the separation of secondary metabolites in EHA from G. max seeds by HPLC were those of factors X1 and X2, using water and EtOH as mobile phase.