Cholesterol with high levels in blood vessels can cause atherosclerosis, stroke, and sudden heart attack. Isolates soy protein have bioactive peptides that have the potential as anticholesterol. This research aims to determine the optimum conditions of hydrolysis and characterize bioactive peptides from soy protein isolate hydrolysates by the bromelain enzyme. Hydrolysis optimization conducted using the enzyme bromelain with levels of 0,2%; and 0,5% (w/v) and variation in incubation time 0, 1, 2, 3, 4, 5 to 6 hours at 45, 50 and 55°C. Protein hydrolysates analyzed the degree of hydrolysis (% DH) and tested anticholesterol activity test through HMG-CoA reductase inhibition test with pravastatin as a positive control. The results showed the optimum conditions hydrolysis of isolate soy protein were obtained at 2 hours, temperature 45 °C with enzyme concentration 0,5% that is by DH value of 40,22%. The highest anticholesterol activity was obtained from the hydrolysate with percent inhibition value of 82,80% (7.371 ppm). SDS-PAGE analysis results show the appearance of bands under 10 KDa and the results of fractionation of bioactive peptide fragments has a molecular weight of 2779 and 2609 Da.