The role of physical and chemical characteristics of biomass has been extensively studied in relation to production of bioethanol and other products. Equally important, however, is the role of biomass processing prior to pretreatment. An economically feasible process must fully utilize biomass from different sources in order to maximize product yields, regardless of factors such as varied moisture content and presence of impurities like bark. We examined the effect of including bark with hybrid poplar, as well as the effect of pre-soaking switchgrass and sugar cane bagasse with water prior to steam explosion pretreatment. In general, pre-soaking improved both overall sugar recovery and enzymatic digestibility. SO2-catalysed soaked switchgrass steam exploded at 205°C for 7.5 min provided 100% cellulose-glucose conversion after 24 hours of hydrolysis at 10% consistency and enzyme loading of 5 FPU/g cellulose. Unsoaked switchgrass treated under the same conditions provided only 75% conversion. It was also shown that the presence of bark does not have a negative effect on enzymatic digestibility. These results show potential to “boost” the effectiveness of existing pretreatment conditions to maximize the yield from a given biomass source. Analytical techniques are an important component to more efficient processing of diverse biomass. On-line monitoring of carbohydrate, ethanol and by-product consumption and formation was studied using Raman spectroscopy. Detection and quantification was possible for many compounds commonly analyzed during bioethanol production, enabling the potential for development of sensors and key points in processing to evaluate composition of various process streams and to improve overall product yields.