The calculation of conversion yields during pretreatment, enzymatic hydrolysis and fermentation of lignocellulosic biomass slurries requires an accurate determination of the fraction insoluble solids (FIS). In previous work (Vicari et al., 2010) we modeled the propagation of uncertainty in the FIS measurement on various lignocellulosic biochemical conversion processes. The results indicate that above a 0.5% relative uncertainty in the FIS measurement, the FIS dominates the uncertainty in monomeric glucose yield from enzymatic cellulose hydrolysis and ethanol yield from sugars during fermentation. Additionally, previous experimental work (Weiss et al., Appl. Biochem. Biotech., 2009) indicates that the uncertainty in FIS measurements is approximately 3% for pretreated corn stover. This work examined the experimental sources of uncertainty in the FIS measurement. The variables examined were sample heterogeneity, pretreatment severity, and total solids content as well as the overall accuracy of the FIS measurement. The results highlight the relative importance of the three variables on FIS measurements and suggest ways to minimize the contribution of sampling errors on FIS measurements. These results will enable the reduction of uncertainty in calculated conversion yields for the various processing steps, and thus the uncertainty in calculated economic measures.