Tanzania Journal of Agricultural Sciences 2013, Vol. 12(2) : 1-9This study describes the effect of high temperature processing of milk and its susceptibility to proteolysis during storage. Raw milk was subjected to various Temperature-time combinations (110, 120, 130 and 142 ˚C for 2s) to assess its susceptibility to proteolysis by native enzymes during storage at 37 ˚C for 28 days. Raw milk and processed milk at low temperature (85/15 s) were also used as benchmarks for the native state and mild heat treatment. Gel electrophoresis (for qualitative analysis) and fluorescamine (for quantitative analysis) methods were used to detect proteolysis in high temperature heated milk caused by native enzymes. For the fluorescamine method, clarification was achieved by isoelectric precipitation and precipitation with acid to obtain pH 4.6 and 6% TCA soluble extracts respectively. Non-clarified samples were used for gel electrophoresis. Both methods confirmed that raw milk and milk processed at 85/15s were the most proteolysed, indicating that the high temperatures (110, 120, 130 and 142 ˚C for 2s) lowered proteolysis through inactivation of heat resistant native enzymes possibly plasmin and hence decreased milk’s susceptibility to spoilage.