The study aimed to investigate the effect of ascorbic acid and soybean protein hydrolysate to iron binding capacity. BPPT (Agency for the Assessment and Application of Technology) developed soybean hydrolysate, which was obtained by thermal and enzymatic hydrolysis. Ascorbic acid was added in sample preparation with composition 0%; 50%; 75% and 100%. Soybean protein hydrolysate was characterized by gel filtration chromatography using sephadex G-25 to separate peptide based on molecular weight. Characterization of soybean protein hydrolysate and fraction hyrdolysate was also conducted, such as protein concentration, molecular weight, iron binding capacity, and amino acids concentration. Iron binding capacity was determined by colorimetric method using ortho-phenanthroline reagent. The result showed that soybean protein hydrolysate increase iron binding capacity 25-37 times than ascorbic acid. Aspartic acid and glutamic acid was found as amino acids that responsible to iron binding capacity in soybean protein hydrolysate.