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 oC), while in the second step, hot-water treatment is used for xylan removal at higher temperatures (150~210 oC). Under optimal conditions (190 oC 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 oC, 12 h reaction time, solid-to-liquid ratio of 1:12 in the first step; and 5.0 ml H2SO4/min, 170 oC, 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 H2SO4) 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).