The search for alternative renewable fuels as replacements for petroleum products is proceeding rapidly. Conversion of plant biomass to fuel is one an attractive source of renewable energy. Among the various sources of biomass, Hybrid poplar (Populus nigra x Populus maximowiczii) is of interest due to its wide availability, high density that reduces cost of transport and improves storabilty and for its high glucan (~ 45%) and xylan (~ 18%) content. Two batches of poplar are being studied in our laboratory containing high lignin (29%) and lower lignin (21%) levels. We present the results of different ammonia fiber explosion (AFEX) pretreatment conditions for both the batches of poplar, including some modifications such as autoclaving the poplar sample prior to AFEX and adding calcium oxide during the AFEX process, followed by enzymatic hydrolysis. We will also discuss additional, inexpensive preprocessing approaches that improve the hydrolysis for pre- and post-AFEX treated samples. Furthermore, optimal combinations of commercially available cellulases and hemicellulases with additives such as surfactants to achieve maximum sugar yields will be discussed. This research is performed as part of the Consortium for Applied Fundamentals and Innovation (CAFI) biomass conversion project.