Thermolysin was used to hydrolyze bovine α-lactalbumin at 25 and 70 °C under non-reducing conditions. The breakdown products were identified by mass spectrometry. At 25 °C, the low proportion of α-lactalbumin in an unfolded state in equilibrium with the native state underwent limited hydrolysis leading to the production of peptides, that were no longer degraded (final peptides). At 70 °C, the protein was in a molten globule-like state according to circular dichroism and complete cleavage of the protein was achieved. At 70 °C, the protein was first quickly cleaved, then unfolded, leading to the release of intermediate peptides, from which final peptides were eventually produced. The amino-terminal 1–58 and carboxy-terminal 95–123 regions were readily cleaved, whereas the central region including the calcium-binding domain was more resistant. Some peptides were produced at 70 °C, but not at 25 °C. The choice of accurate experimental conditions may be of importance for the preparation of functional peptides.