Disclosed is a method for evaluating in vivo protein nutrition based on an LC-MS-MS technique, including the following steps: (1) collecting contents from different intestinal segments, and extracting and isolating protein ingredients; (2) determining the concentration of proteins; (3) treating before carrying out mass spectrometry: including digestion and desalting of a whole protein solution; (4) LC-MS-MS analysis; (5) database searching; and (6) data processing. Proteomic technology is used to identify proteins in the contents of different intestinal segments and digestive products thereof, and the source of the proteins in the contents of different intestinal segments and the contents thereof can be determined therefrom. Through bioinformatic analysis, the function of differential proteins in the body can be further understood, where the gene expression of enzymes related to protein digestion and metabolism may be different, thereby providing a scientific basis for further scientific evaluation of protein digestion and utilization.