We found that fructose causes a dramatic decrease in the tolerance level and survival rate of all the yeasts tested, Saccharomyces and non-Saccharomyces, a finding that to our knowledge has not been reported before, as usually tolerance/resistance determinations in the literature are carried out using solely glucose as the carbon source. Accordingly, there is a high risk of overestimation of ethanol tolerance of a given strain when tested in the laboratory using only glucose, when it is intended for performing fermentation in fructose-rich substrates. The methodology and analysis procedure proposed here for stress tolerance quantification applied to several genera of yeasts is proposed for screening and selection of those strains with a high performance on fermentation processes, but also could be used as a tool for isolation of those variants/segregants of a yeast/bacteria population with a more resistant phenotype, allowing a direct calculation of the ratio of this subpopulation from the original sample