Donkey’s milk may be considered a valid alternative, for infant nutrition, to powdered milks, soybean milk or other formulas, since its composition in lipids and proteins is very close to human milk. The aim of the present study is to characterize “donkey’s milk” product, especially with regard to the protein fractions. Three different chromatographic approaches for donkey’s milk proteins separation are described, based on their different isoelectric points, hydrophobicity and molecular mass. The proteins obtained were further separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis and identified by N-terminal sequencing. αS1- and β-caseins, lysozyme, α-lactalbumin, β-lactoglobulin were identified. Furthermore, the content of α-lactalbumin, β-lactoglobulin, and lysozyme in donkey’s milk was determined by a reversed-phase HPLC method. The β-lactoglobulin and α-lactalbumin content was 3.75 and 1.8 mg/ml, respectively (mean values). The present study showed the nutritional properties of donkey’s milk characterized by low casein and high lysozyme content (1.0 mg/ml) compared with other kinds of milks.