Donkey milk has been recently revalued intensely due to its nutritional properties. Moreover, donkey milk has been proposed as an effective alternative food for some infants with cow milk allergy. Two fast analytical methods were proposed to detect the fraudulent practice of blending cow milk to donkey milk. Detection of cow αs1-casein bands along the profiles of experimental donkey–cow milk mixtures analyzed by isoelectric focusing was adequate to estimate cow milk used as adulterant of donkey milk starting from 5% (v/v). An ELISA-based method using the antipeptide antibodies raised against the 1–28 sequence stretch of cow β-casein was also developed for an accurate definition of composition of donkey–cow milk mixtures. The presence of cow milk at levels as low as 0.5% (v/v) was detected in donkey–cow milk mixtures prepared at laboratory scale and assayed by ELISA.