Galactooligosaccharides (GOS) synthesis, carbohydrates with an unit of glucose and n units of galactose, is performed by the reaction called transgalactosylation, conducted through the enzyme β-galactosidase. The use of by-products from food industry, such as whey permeate, is sustainably desirable. The aim of this work was to compare enzymatic GOS production in lactose analytical solution and whey permeate solution. Among the materials used are: β-galactosidase enzyme from Aspergillus oryzae, lactose monohydrate grade P.A., whey permeate powder, rotary shaker and HPAED-PAD (Dionex). A solution containing 10U of enzyme was incubated into two types of solutions, the first containing 30% (w/w) lactose, and the second containing 34.7% (w/w) whey permeate, both in citrate-phosphate buffer pH 4.5, at 45°C in a rotary shaker at 200 rpm. Products obtained were analyzed by Anion Exchange Chromatography on HPAED - PAD equipment, using 16mM NaOH 50% (w/w) solution as the mobile phase and Dionex PA1 column. Results show a of 35.02 g/L and 57.61 g/L GOS production, with a 15% and 29% hydrolysis degree in lactose and whey permeate media respectively. According to the data obtained, there was a stabilization tendency in GOS production due to the observed increase in glucose concentration, inhibitor of the enzymatic process. GOS production in Whey permeate was higher than that found in lactose medium, indicating that the composition of whey medium, rich in minerals and small peptides, is suitable for obtaining oligosaccharides.