Abstract: Bovine serum albumin and human urinary protein gel bands of two different molecular weight were digested by 20 W, 100 W and 200 W ultrasound-assisted in-gel digestion method, and the digested peptides were analyzed by high-resolution mass spectrometry. By comparing the number of identified proteins and peptides, the optimal ultrasonic power for ultrasound-assisted in-gel digestion was determined. Meanwhile, the peptide characteristics of ultrasound-assisted in-gel digestion method were analyzed by comparing with traditional overnight digestion method. 20 W ultrasound-assisted in-gel digestion method shows the minimal gel damage and loss for in-gel protein samples, the number of peptides identified in BSA is 65, while the number of identified peptides from 100 W, 200 W and the traditional overnight digestion methods is 57, 51 and 41, respectively, much lower than 20 W ultrasound-assisted in-gel digestion method. The number of proteins identified in two different gel bands of human urinary protein in 20 W ultrasound-assisted in-gel digestion method is 168 and 199, respectively, which is better than 100 W, 200 W and the traditional overnight digestion method. By analyzing the mis-cleavage rate and semi-tryptic rate of peptides based on the qualitative and quantitative analysis, the mis-cleavage rate of ultrasound-assistedin-gel digestion is higher than traditional overnight digestion method, while the semi-tryptic rate of ultrasound-assisted in-gel digestion is lower. The mis-cleavage type analysis of identified peptides indicates that traditional overnight method can generate more Proline (P) type mis-cleavage peptides, and ultrasound-assisted in-gel digestion method produces more aspartate/glutamate (D/E) type mis-cleavage peptides. The mis-cleavage peptide types from three ultrasound powers show no significant differences. Compared with traditional overnight digestion method, ultrasound-assisted in-gel digestion method can significantly shorten in-gel protein digestion-time and 20 W ultrasonic power shows the best digestion results compared with 100 W ultrasonic power and 200 W ultrasonic power. Furthermore, ultrasound assisted in-gel digestion method can obtain more protein/peptide identifications. The peptide mis-cleavage rate in ultrasound-assisted in-gel digestion method is higher than traditional overnight digestion method, while the semi-tryptic rate is lower. Above results will benefit the application of ultrasound-assisted in-gel digestion method for proteomic analysis.