Soybeans contain proteins that have the potential to produce anticancer bioactive peptides. This study aims to determine the anticancer activity of soy protein hydrolysate against MCF-7 breast cancer cells. Soybean protein hydrolyzed by bromelain enzyme 0.5% (w/v) at the optimum temperature and pH for protein hydrolysis using the Bergmeyer and Grassl method. The degree of hydrolysis value of protein hydrolysate was determined by the Alder-Niesen method and the protein profile was analyzed by SDS-PAGE. The hydrolysate with the best degree of hydrolysis value was analyzed for anticancer activity against MCF-7 breast cancer cells by the Presto Blue assay method, and fractionation of protein hydrolysates by gel filtration chromatography (Sephadex G-15). The molecular weight of the peptide was characterized by LCMS/MS. Soy protein hydrolysis using 0.5% (w/v) bromelain enzyme was optimum at 65 ºC and pH 7.0 for 4 hours, with a hydrolysis degree value of 20.57%. The SDS-PAGE analysis showed that the protein hydrolysates had quite thick protein bands in the range of <35 kDa with an IC50 value of 70.37 mg/mL. Based on the LCMS/MS results, the peptide from fractionation has a molecular weight of 5.133 kDa.