This chapter focuses on the four types of pretreatment routes: physical, chemical, physico-chemical, and biological. It also includes initial biomass processing consisting of harvesting, transport, and storage. Lignocellulosic biomass consists of cellulose, hemicellulose, lignin, and other minor components. Cellulose and hemicellulose are polymers of sugars that can be converted into sugar products. Pretreatment of lignocellulosic natural fibers is a critical step in the cellulosic biomass-to-biofuel conversion as it has the ability to increase yield and decrease process costs. Storage allows for continuity in feedstock supply to the biorefinery without putting strain in the cultivation and harvesting process. Milling reduces the size of the biomass which makes it susceptible to enzymatic hydrolysis. Irradiation applies a uniform heat to lignocellulosic biomass at a very short time. Hydrothermal pretreatment employing subcritical water has attracted much attention because of its suitability as a non-toxic, environmentally benign and inexpensive media for chemical reactions.