In the present work we report a novel on-target protein cleavage method. The method utilizes ultrasonic energy and allows up to 20 samples to be cleaved in 5 minutes for protein identification and 1 sample in 30 s for on-tissue digestion. The standard proteins were spotted on a conductive glass slide in a volume of 0.5 μl followed by 5 minutes of ultrasonication after trypsin addition. Controls (5 min, 37°C no ultrasonication) were also assayed. For the tissues, after trypsin addition, digestion was enhanced by 30 s of ultrasonication. The samples were analyzed and compared to those obtained by using conventional 3-h heating proteolysis. The low sample volume needed for the digestion and the reduction in sample handling steps and time are the features that makes this method appealing to the many laboratories working with high-throughput sample treatment.