Cow’s milk protein allergy (CMPA) is an abnormal immunological response to cow milk proteins, which results in IgE-mediated reactions. The therapeutic strategy to respond to CMPA envisages the total elimination of milk or the administration of cow’s milk substitutes. For this reason, the use of milk from other mammalian species was tested. Among them, donkey’s milk proved to be the best alternative in feeding infants affected by CMPA, since it's chemical composition is comparable to human milk. In this work, an in vitro study was performed in order to analyze the IgE reactivity to milk protein allergens from cow, donkey, and goat. In particular, immunoblotting experiments using sera from milk-allergic and non-allergic adult volunteers were conducted with the aim of verifying the hypoallergenic property of donkey’s milk. This study provided preliminary evidence of the hypoallergenicity of donkey’s milk when compared to bovine and goat milk. Considering the obtained results, it would be possible to develop a sensitive diagnostic method for CMPA detection, based on the chromatographic and immunoblotting analysis.