Abstract: OBJECTIVE To accurately and efficiently detect the horse hide components in Asini Corii Colla (ACC) by using ultrasonic assisted fast enzyme digestion and tandem mass spectrometry， and provide experimental basis for ACC quality standard investigation. METHODS  By using ultrasonic assisted trypsin digestion method， ACC hydrolysate samples were prepared rapidly. High performance liquid chromatography with three quadrupole mass spectrometry was used to detect the ACC hydrolysate samples. Under positive electrospray ionization mode and multiple reaction monitoring (MRM)， donkey-specific peptide (Pep-1) with ion pair of m/z 691.0 → 809.4 and horse-specific peptide (Pep-2) with ion pair of m/z 698.3 → 809.4 were used to detect Pep-2 in ACC， and the relative peak area ratio between Pep-2 and Pep-1. RESULTS This method could be used to determine if there was horse-hide gelatin (HHG) in ACC within 20 min， and the proportion of HHG in ACC. The detection limit of HHG adulteration was low to 1%. CONCLUSION Ultrasound-assisted fast enzyme digestion combined with tandem mass spectrometry showed good accuracy， reliability， specificity and efficiency， and can be used for the rapid detection of HHG in ACC.