Sterilization and UHT treatment are commonly used to increase the shelf life of milk and dairy beverages. However, the high heat resistance of the plasmin system enables it to partially survive UHT treatment, which could lead to defects in the texture (gelation, sedimentation) and the taste (bitterness) of these products. The present study investigated the effect of high heat treatment on plasmin-induced hydrolysis using a sequential approach. A high-heat-treated (120°C/15 min) skim milk had been found to have higher resistance to plasmin-induced hydrolysis, compared with a control skim milk. The effect of heat treatment on the resistance of skim milk was studied with a sequential approach. Different milk systems were prepared by removing whey protein and then lactose from skim milk and reconstituting each milk separately, to observe the effect of whey protein and lactose both individually and in combination. The milk systems were heated at 120°C for different times to achieve different levels of heat treatment. All treated milk systems were then hydrolyzed by adding plasmin and the reaction was monitored using reverse-phase HPLC. The extents of whey protein denaturation, dissociation of casein from the micelles and association of whey protein with the micelles were monitored using sodium dodecyl sulfate-PAGE. Particle size and levels of lactosylation were also measured. Plasmin-induced hydrolysis was negatively affected by an increase in the heat treatment. Both whey protein association with the casein micelles and lactosylation decreased the availability of protein to plasmin. These effects were compared and mechanisms for them have been proposed. Whey-protein-free milk was the most resistant to plasmin, followed by skim milk and lactose-free milk, suggesting that lactosylation plays a more major role than whey protein association with the casein micelles in making protein resistant to plasmin-induced hydrolysis.