Whey, a protein-rich by-product of cheese production, offers nutritional and bioactive potential. Donkey milk whey (DW) may serve as an innovative protein source, yet studies on its properties remain limited. This study evaluated DW for chemical composition, antioxidant activity, and changes in protein digestibility during simulated in vitro digestion using the INFOGEST protocol. Proteolytic activity mediated by digestive enzymes was assessed through Kjeldahl, Bradford, and SDS-PAGE methods, while peptide hydrophobicity was evaluated via RP-HPLC. DW was found to have a moisture content of 91.88 g/100 mL, ash content of 0.45 g/100 mL, reducing sugars content of 3.47 g/100 mL, pH 3.5, and acidity of 0.24 g/100 mL. Total protein in DW was 1.27 g/100 mL, decreasing to 0.93 g/100 mL in the Gastric Digest (GD) and 0.73 g/100 mL in the Intestinal Digest (ID), reflecting a digestibility of 41.43%. Soluble protein content in DW was 0.55 g/100 mL, reducing to 0.03 g/100 mL in GD and ID, indicating a high soluble protein digestibility (94.54%). SDS-PAGE confirmed protein hydrolysis by digestive enzymes, while RP-HPLC revealed hydrophilic peptides formation with progressive digestion. DW chromatogram exhibited a total area of 22.16 x 10⁴, which decreased in GD (21.54 x 10⁴) and ID (17.70 x 10⁴), indicating protein degradation with new smaller peaks post-digestion. Antioxidant capacity (ABTS and FRAP) was highest post-intestinal digestion, with mean values of 182.67 µmol TE/100 mL and 52.84 µmol TE/100 mL, respectively. These findings indicate high protein digestibility in DW, producing more hydrophilic and potentially bioactive peptides with enhanced antioxidant activity post-digestion.