AbstractFreshly harvested alfalfa was fractionated using centrifugation and filtration, whereby alfalfa was separated into a fiber-rich cake and a nutrient-rich juice. The solid cakes from the above separation processes were used as the feedstock for ethanol production using separate hydrolysis and fermentation. The filtration process proved to be more efficient at reducing the solids mass transfer to the juice than the centrifuge process. Glucose from filtered alfalfa solid cake can be efficiently fermented to ethanol with 75% of the theoretical yield. In conclusion, centrifugation was not as effective as filtration in removing particulates and colloidal matter from alfalfa. The filtration process resulted in a solid cake with a higher cellulose digestibility, which leads to a higher ethanol production.