Trehalose is a non-reducing disaccharide consisting of two glucose units linked to anomeric carbon, present in several organisms, which plays a protective role when yeasts are under physiological stress conditions. Its synthesis occurs in response to some type of stress faces by the body, such as dehydration, heat stress, oxidative stress, osmotic stress and nutritional stress. It plays an important role in maintaining the integrity of the cell membrane, acting to protect the cell’s cytosol components from adverse conditions. The understanding of trehalose metabolism by yeasts is linked to their use for the production of fuel ethanol, since high concentrations of ethanol and sugar in the environment in which the yeasts are inserted are stress factors. Therefore, the understanding of trehalose synthesis by the yeasts under these conditions is important to assess their cell viability in high gravity fermentations, with high levels of reducing sugars, in order to find alternatives to increase ethanol yield and productivity by industries. As a result, the objective of this work was to verify the effect of osmotic stress on trehalose accumulation. The cultivation of (PEDRA-2) yeasts was carried out in a 250 mL Erlenmeyer flask containing 50 mL of YPD (1% Yeast Extract, 2% Peptone, 2% Dextrose) added with the concentrations of total reducing sugars (TRS) of 40, 45 and 50% being inoculated 1 mL of rehydrated yeast, being coupled to devices for online monitoring of biomass (CGQ-Cell Growth Quantifier) under stirring at 150 rpm for 5 hours, later, the cultures were centrifuged and washed with distilled water to remove glucose and sugars in the middle. Trehalose was extracted from 12 mg (dry weight) of cells with 0.5M trichloroacetic acid and determined by the Antrone method. The results were submitted to Tukey’s test (p<0.05), being possible to observe that with 40% of TRS accumulated 133.4 µg/100g yeast, 45% TRS accumulated 163.1 µg/100g yeast and 50% TRS accumulated 179.7 µ/100g of yeast. It has been found that the increase in sugar concentration stimulates the yeast to bioaccumulate trehalose to support osmotic stress.