Lignocellulosic biomass is a potential feedstock for bioethanol production. Biomass hydrolysates, prepared with a procedure including pretreatment and hydrolysis, are considered to be used as fermentation media for microorganisms, such as yeast. During the hydrolysate preparation procedure, toxic compounds are released or formed which may inhibit the growth of the microorganism and thus the product formation. To study the effects of these compounds on fermentation performance, the production of various hydrolysates with diverse inhibitory effects is of importance. A platform of methods that generates hydrolysates through four different ways and tests their inhibitory effects using Bioscreen C Analyzer growth tests is described here. The four methods, based on concentrated acid, dilute acid, mild alkaline and alkaline/oxidative conditions, were used to prepare hydrolysates from six different biomass sources. The resulting 24 hydrolysates showed great diversity on growth rate in Bioscreen C Analyzer growth tests. The approach allows the prediction of a specific hydrolysate's performance and helps to select biomass type and hydrolysate preparation method for a specific production strain, or vice versa.