− A two-step process was investigated for pretreatment and fractionation of rice straw. The two-step fractionation process involves first, soaking rice straw in aqueous ammonia (SAA) in a batch reactor to recover lignin-rich hydrolysate. This is followed by a second-step treatment in a fixed-bed flow-through column reactor to recover xylooligomer-rich hydrolysate. The remaining glucan-rich solid cake is then subjected to an enzymatic process. In the first variant, SAA treatment in the first step dissolves lignin at moderate temperature (60 and 80 o C), while in the second step, hot-water treatment is used for xylan removal at higher temperatures (150~210 o C). Under optimal conditions (190 o C reaction temperature, 30 min reaction time, 5.0 ml/min flow rate, and 2.3 MPa reaction pressure), the SAA-hot-water fractionation removed 79.2% of the lignin and 63.4% of the xylan. In the second variant, SAA was followed by treatment with dilute sulfuric acid. With this process, optimal treatment conditions for effective fractionation of xylo-oligomer were found to be 80 o C, 12 h reaction time, solid-to-liquid ratio of 1:12 in the first step; and 5.0 ml H 2 SO 4 /min, 170 o C, and 2.3 MPa in the second step. After this two-step fractionation process, 85.4% lignin removal and 78.9% xylan removal (26.8% xylan recovery) were achieved. Use of the optimized second variant of the two-step fractionation process (SAA and H 2 SO 4 ) resulted in enhanced enzymatic digestibility of the treated solid (99% glucan digestibility) with 15 FPU (filter paper unit) of CTec2 (cellulase)/g-glucan of enzyme loading, which was higher than 92% in the twostep fractionation process (SAA and hot-water).