AbstractWater hyacinth was analyzed to determine its hemicellulose/lignin content, evaluating the conditions for the saccharification process with commercial microbial enzymes. Plant material, including leaves and stalks, was pretreated at several temperatures (100, 110, and 120 °C) with different sulfuric acid concentrations (0.5, 1.0, 1.5, 2.0, 2.5, and 3%) and residence times (0, 15, 30, 45, 60, 90, and 120 min). Total reducing sugars were measured by the dinitrosalicylic acid method. The optimum conditions that maximized the yield of reducing sugars included a pretreatment with 2% (v/v) sulfuric acid at 110 °C for 90 min. The optimum conditions for enzymatic saccharification used the commercial enzyme Celluclast at 50 °C for 24 h of hydrolysis. The maximum yield was 0.54 g of fermentable sugars per gram of biomass. Data demonstrated that E. crassipes is suitable as a raw material for products such as bioethanol; however, further fermentation studies are required.