This study aimed to evaluate the properties of RS 3 prebiotic from modified arrowroot starch, a result of in-vitro combination of acid, enzymatic and physical modification. Arrowroot starch resulting from combination between acid hydrolysis (2.2 N HCl, 2 hrs), debranching using enzymes pulullanase (10.4 U/g starch), 3 cycles of autoclaving-cooling (121ºC, 15 min), and heat moisture treatment (121ºC, 1 hr), has increased level of resistant starch. The evaluation of the prebiotic properties of RS 3 in modified arrowroot starch was performed in vitro. Modified arrowroot starch from a combination of hydrolysis acid (AH), debranching (D), and autoclaving-cooling (AC) and combinations of acid hydrolysis, debranching, autoclaving-cooling, and heat moisture treatment (AHDAC-HMT) modification process had the highest resistance to artificial gastric acid at more than 96% in pH 2-4 after 2 hrs. Arrowroot starch from AHDAC and AHDAC-HMT modification promoted the growth of probiotic bacteria Lactobacillus acidophilus and significantly affected its prebiotic index with above 2.0 value. However, only the arrowroot starch with AHDAC combination modification suppressed the growth of pathogenic bacteria. It can be concluded that arrowroot starch modified with AHDAC is a potential source of prebiotic for its resistance against artificial gastric acid with good prebiotic properties.