The development of protein hydrolysates as a source of peptides has attracted the attention of the food and pharmaceutical industries due to their functional and bioactive properties. Several studies highlight the potential of bioactive peptides derived from the hydrolysis of whey proteins, but studies with donkey whey hydrolysates are scarce. This study evaluated the soluble proteins, the protein profile by SDS-PAGE, the hydrophobicity profile by RP-HPLC, the tryptophan content and the antioxidant activity (ABTS and FRAP) of DW and whey hydrolysates obtained by enzymatic hydrolysis from Alcalase (DWA) and Pepsin (DWP). The soluble protein content in DW was 0.56 g/100mL, with a tryptophan content of 24.06 mg/100mL. Hydrolysis with pepsin (DWP) reduced the soluble proteins to 0.03 g/100mL, while the tryptophan content increased to 30.64 mg/100mL. DWA presented 0.09 g/100mL of soluble proteins and the highest tryptophan content (39.46 mg/100mL). DW presented antioxidant activity of 95.18 µmol ET/100mL (ABTS) and 33.84 µmol ET/100mL (FRAP). The antioxidant activity increased in the hydrolysates, reaching 147.2 µmol ET/100mL (ABTS) and 48.83 µmol ET/100mL (FRAP) in DWP. DWA showed the highest antioxidant activity, with values of 186.66 µmol ET/100mL (ABTS) and 62.65 µmol ET/100mL (FRAP), indicating that Alcalase was even more efficient, possibly releasing smaller peptides with greater antioxidant capacity. These results are confirmed in SDS-PAGE electrophoresis, where disappearance of protein bands was observed in DW after hydrolysis. The hydrophobicity profile showed an increase in the number of peaks in the low hydrophobicity zone and an increase in the total area in the hydrolysates, confirming the release of more hydrophilic peptides in DWP and DWA. The study highlights the potential of donkey whey hydrolysates as a source of potentially bioactive low-hydrophobic peptides, associated with increased antioxidant activity after hydrolysis.