Ultrasound assisted extraction process (UE) was employed to extract the betalain from Malabar spinach fruit using response surface methodology (RSM). Extraction process variables such as temperature, mass of the sample, extraction time was investigated using Box-Behnken design (BBD). Quadratic polynomial model was developed. Numerical optimization shows that sample mass of 9 g, temperature of 35°C and extraction time of 18 min, were yield maximum betalain. Under these conditions, 18 mg of betalain was extracted.