Cotton stalk is a holocellulose rich, inexpensive agricultural residue available in surplus without any competitive uses neither as food nor as animal fodder. These aspectshold high potential for cotton stalk as a biomass to be suitable for ethanol production. Dilute acid pretreatment conditions on bench scale have been optimized for cotton stalk by Response Surface Methodology (RSM) using Central Composite Design (CCD). Effect of four pretreatment process variables viz. temperature, acid concentration, time of reaction and stirring speed has been optimized for maximum enzymatic sugar release during the subsequent enzymatic saccharification. Under the optimized pretreatment conditions, i.e., temperature: 157. °C, acid concentration: 1.07% (w/w),and time: 20 min, enzymatic sugar releasewas found to be 684 mg/g of dry pretreated biomass. A correlation of hemicellulose removal and inhibitor formation with combined severity factor (CSF) was drawn. Mass balance carried out for the pretreatment step under optimized conditions resulted in 68.35 and 8.31% of xylose and glucose saccharificationyieldsrespectively. Subsequent enzymatic saccharification yieldsofglucose and xylose were 93.56 and 19.93% respectively. The overall saccharification yield integrating pretreatment and enzymatic hydrolysis of cotton stalk was 91.06%. Physicochemical characterization of native and pretreated biomass was carried out by compositional analysis, FT-IR and XRD revealing significant changes in biomassproperties responsible for improved saccharification efficiency.