Kunitz inhibitor (KTI) and Bowman-Birk trypsin inhibitor (BBI) are inhibitors of digestive enzymes in raw soybeans. Due to their antinutritive properties in the active state, their inactivation by heat treatment is commonly used. Soymilk is a turbid and stable colloidal solution, obtained by thermal treatment of soybean. In this study soymilk was made on a pilot-plant scale from six soybean cultivars using hydrothermal cooking (HTC) as the production method. This procedure is significantly different from the traditional one. The aim of this investigation was to evaluate the impact of the HTC processing for soymilk production and different soybean genotypes on trypsin inhibitor content and activity. Obtained soymilk contained BBIin trace amounts, in the BBI-polymeric forms. The BBI monomeric forms were not detected. The soymilk of the investigated soybean genotypes had very similar KTI levels (2.34–2.99%). Results have suggested that the soybean genotype does not have substantial effects on the levels of KTI, as well as on the value of residual trypsin inhibitor activity (rTIA). The total content of TI and rTIA showed a strong dependence (r=0.91; p<0.05). HTC-soymilk rTIA was <20% (7.15–19.89%). These results have indicated that HTC processed soymilk is applicable for human consumption.