In this work, the viability of using aqueous-phase sugars derived from the pyrolysis of lignocellulosic biomass was analyzed using high-throughput screening in microtiter plates. To standardize results, a synthetic aqueous phase of pyrolytic bio-oil was constructed based on typical constituents and composition ranges and then used to determine the fermentation viability of Saccharomyces cerevisiae. The effects of inhibitory compounds in pyrolytic bio-oil were assessed by fitting measured growth kinetics to the model of Baranyi and Roberts (Int. J. Food Microbiol. 1994, 23, 277), specifically on the fitted growth rates, initial microorganism adaptation, and maximum biomass densities. It was found that even a dilution to approximately 10% of the hypothetical inhibitor concentration in aqueous bio-oil was significantly inhibitory to growth, although the presence of additional sugars was able to moderate this impact slightly. The high-throughput screening used in this work allowed for the rapid measurement of a variety of inhibitors at different concentrations, as well as inhibitory mixtures.