The need to replace fossil fuels has motivated research to develop alternative technologies for renewable energy production. For second generation ethanol, this includes the high-throughput screening and selection of yeast strains resistant to inhibitory byproducts of biomass pretreatment. In this work, we evaluated the use of static growth in 96-well microplates as a fermentative assay for Saccharomyces cerevisiae high-throughput screening, together with robust OD measurement correlations that overcome the low absorbance limit found in conventional plate spectrophotometers. This resulted in a method of yeast cultivation in microplates that requires no agitation and results in accurate kinetic parameters that can be used for the screening of S. cerevisiae. In order to automate calculation of these physiological parameters, we created OCHT (R), an open-source high-throughput phenotype profiling software for processing, analyzing and visualizing S. cerevisiae growth data. Together, the cultivation standardization and data analysis toolbox allowed the evaluation and comparison of four S. cerevisiae strains in media containing aldehydes growth inhibitors, typically found in hydrolysates produced from sugarcane biomass. We identified FMY001 (SA1-derived) as a tolerant strain at high concentrations of HMF, furfural and vanillin. The individual analysis of segregants derived from FMY001 demonstrated their potential application in emerging biotechnological processes and gave us preliminary insights about the genetic mechanisms related to its high HMF concentration tolerance.