The Microtox assay represents a rapid, accurate, and reproducible method for determining general microbial toxicity. This assay was used to evaluate the relative toxicity of a variety of hydrolysate samples derived from dilute-acid and alkaline biomass pretreatment. Toxicity is elicited from biomass degradation products, such as furfural, hydroxymethyl furfural, and acetic acid, generated during pretreatment. Microtox results indicate that the pretreatment samples examined ranged from 9 to 71 toxicity units (TU). Correlations of TU and sample absorbance at several wavelengths were evaluated for all sample series. Sample TU values best agreed with absorbance at 230 nn, but the unsatisfactory fit suggests that absorbance should not be used as an absolute measure of sample toxicity. Microtox data for pretreatment hydrolysate samples were correlated with the inhibition experienced by the ethanologenic yeast, Saccharomyces cerevisiae strain D(5)A, during the simultaneous saccharification and fermentation (SSF) process of pretreated biomass. None of the alkaline pretreatment conditions produced inhibition during SSF (data not shown). However, the acid pretreatment conditions did produce a wide range of inhibitory and noninhibitory hydrolysates. In general, fermentation was inhibited for acid-pretreated hydrolysate samples with values exceeding 45 TU. Preliminary studies that focused on reducing hydrolysate sample toxicity (detoxification) indicate that adding perlite and zeolite had little effect. However, the use of charcoal, a universal flocculent, or ion-exchange resins significantly reduced sample toxicity, holding promise for the efficient bioconversion of pretreated biomass to ethanol. Moreover, the developed toxicity measurement assay can quickly monitor the quality of the pretreatment process. In this way, biomass conversion operation processes can be reliably controlled at the pilot and commercial scales.