To date, there is limited knowledge available regarding the key features of pretreated lignocellulosic substrates that promote the effective enzymatic hydrolysis of the cellulose component to glucose during bioconversion processes to produce ethanol. Fundamentally, cellulase enzymes require access to the cellulose to carry out effective hydrolysis. Porosity and the overall surface area of substrates have major structural features influencing the hydrolysis of pretreated substrates by cellulases. Simons' Stain (SS) is a potentially useful semiquantitative method for estimating the available surface area of lignocellulosic substrates. In this study, a modified, rapid SS method was developed, where the processing time was decreased from >50 to 6 h and the maximum dye adsorbed on the substrate was calculated using the adsorption isotherm for the orange and blue components of the dye mixture. The modified SS test readily measures the decrease in accessibility and hydrolyzability of a steam pretreated substrate that had been dried under three different drying regimes. For each of the lignocellulosic substrates, the total dye adsorption correlated well with the hydrolysis yields resulting in a correlation coefficient of r2 = 0.95. The modified SS procedure is an effective tool for assessing how lignocellulosic substrates might be potentially hydrolyzed by cellulases.