Dilute-acid pretreatment will most likely be the initial process in cellulosic biofuels production. Effective acid impregnation of biomass is required to achieve high xylose yields and minimize degradation products during pretreatment, as well as prepare the biomass surface for enzymatic hydrolysis. Biomass neutralization capacity, structure, acid loading, and acid solution saturation affect impregnation efficacy. To compare various impregnation processes, a rapid, accurate, and reproducible method of determining the “in situ” acid concentration in biomass is needed. Previous methods are time consuming and ineffective for low acid concentrations, particularly below 0.6%, because of biogenic acid production. Homogenization, incubation, and refrigeration were the three different methods analyzed to extract acid from impregnated biomass. Homogenization was tested using two different devices, a blender and a homogenizer. We analyzed 16 biomass samples representing 8 different impregnation conditions. The varied factors among each sample condition included the impregnation reactor (spray or soak), acid concentration (0.2 wt% or 1.08 wt%), and particle size ( ¼ ” or ¾”). We found that biogenic acid production at low acid impregnation concentrations (<0.6% H₂SO₄) is exhibited during incubation. A single-factor, 95% confidence ANOVA test showed that homogenization and refrigeration are both suitable methods to determine biomass acid concentration. However, because refrigeration took 48 hours and homogenization took 15 minutes, the latter was found to be the most suitable method of ascertaining biomass acid concentration.