Extraction of steroidal glycoside from small-typed bitter gourd was optimized by central composite design (CCD) considering two factors: extraction temperature (50°C - 150°C) and ethanol concentration (50% - 100%). The optimum extraction conditions were found to be ethanol concentration of 50% and extraction temperature of 150°C. Under the optimized conditions, the experimental value of steroidal glycoside yield was 10.23 mg/50 g dried bitter gourd, which reasonably close to the predicted value (12.03mg.50 g dried bitter gourd). Previous detection method of steroidal glycoside by high performance liquid chromatography (HPLC) was improved and validated. The quantitative determination of steroidal glycoside was performed on a C18 symmetry column with aqueous methanol as mobile phase at a flow rate of 1 mL/min and injection rate of 200 uL. The eluents were examined at 204 nm using photo diode array (PDA) detector. The improved method was validated for its linearity, repeatability, precision, accuracy, limit of detection and limit of quantification and found to be satisfactorily. Extracted steroidal glycoside showed good DPPH free radical scavenging, equivalent to 2.29 g Trolox/100 g dried bitter gourd and 0.63 g gallic acid/100 g dried bitter gourd respectively while its carbohydrate-hydrolyzing enzyme inhibitory effect was comparable to conventional enzyme inhibitor drug, acarbose.