Kunitz trypsin inhibitor (KTI) and Bowman-Birk inhibitor (BBI) have trypsin inhibitor activities (TIA), which could cause pancreatic disease if at a high level. It is not clear why some KTI and BBI lose TIA and some does not in the soymilk processing. This would be examined in this study. TIA assay showed residual TIA was decreased with elevated temperature and TIA was decreased quickly in the beginning and then slowly in boiling water bath. Interestingly, ultracentrifugation showed low residual TIA soymilk had more precipitate than high residual TIA soymilk and soymilk TIA loss had a high correlation coefficient (R(2) > 0.9) with precipitate amount. In addition, the TIAs of floating, supernatant, and precipitate obtained by ultracentrifugation were assayed and >80% residual TIA was concentrated in the supernatant. Tricine-SDS-PAGE showed KTI in supernatant was mainly a noncovalent bound form which might exist as itself and/or incorporated into a small protein aggregate, while KTI in precipitate was incorporated into a protein aggregate by disulfide and/or noncovalent bonds. Chymotrypsin inhibitor activity (CIA) assay showed about 89% of the original CIA remained after 100 °C for 15 min. Ultracentrifugation showed that >90% residual CIA was concentrated in supernatant. Tricine-SDS-PAGE showed soymilk (100 °C, 15 min) BBI mainly existed in supernatant but not in precipitate. It was considered that BBI tended to exist as itself with its natural conformation. Thus, it was suggested residual TIA was mainly from the free BBI and TIA inactivation was mainly from KTI incorporation into protein aggregate. This study is meaningful for a new strategy for low TIA soymilk manufacture based on the consideration of promoting protein aggregate formation.