Abstract: Trypsin specifically cleaves peptide bonds at arginine and lysine residues, making it the most frequently utilized specific endopeptidase in proteomics and protein quantification studies owing to its high cleavage efficiency, strong specificity, and wide applicability in various experimental systems. However, during proteolytic digestion, it often produces non-specific cleavage at sites containing phenylalanine (F), tyrosine (Y), and tryptophan (W), which may interfere with subsequent peptide separation and identification, reduce the reliability of experimental results, and even lead to misinterpretation of protein expression profiles. To explore the characteristics and intrinsic causes of trypsin’s non-specific cleavage, as well as the specific impacts of this phenomenon on the accuracy of protein analysis, this study systematically evaluated the cleavage specificity of trypsin derived from different sources and subjected to different methods. The trypsin reagents included conventional trypsin derived from bovine pancreas that had been treated with tosylphenylalanyl chloromethyl ketone (TPCK) to inactivate chymotrypsin; mass spectrometry sequencing-grade trypsin, also derived from bovine pancreas, which was not only treated with TPCK for chymotrypsin inactivation but also modified via methylation to enhance its stability and cleavage specificity; and recombinant trypsin expressed in eukaryotic systems, which can effectively eliminate contamination by other proteases. Meanwhile, this study comprehensively investigated the effects of key digestion parameters, including substrate concentration, enzyme-to-substrate ratio, reaction pH, reaction temperature, and reaction duration, on non-specific cleavage to identify the key factors of this phenomenon and provide targeted optimization strategies. The results showed that the source and processing methods of trypsin are key factors affecting the levels of non-specific cleavage. Conventional trypsin has obvious chymotrypsin contamination and a strong autolysis tendency, which directly leads to a markedly higher level of non-specific cleavage compared with sequencing-grade and recombinant trypsin. In addition, enzyme concentration displays a significant positive correlation with the non-specific cleavage rate, and alkaline conditions can further intensify this non-specific cleavage phenomenon. Conventional trypsin reaches maximum cleavage efficiency at 37 ℃; its non-specific cleavage rate is inversely proportional to substrate concentration and rises progressively with the extension of reaction time. In contrast, sequencing-grade and recombinant trypsin maintain stable cleavage specificity against variations in temperature, reaction time, and substrate concentration, with their non-specific cleavage rate remaining at a low level. In summary, prioritizing the use of high-purity modified trypsin and scientifically optimizing enzymatic digestion parameters during experiments can effectively mitigate non-specific cleavage, ensure the accuracy and reliability of protein analysis results, and establish a solid foundation for subsequent proteomic research, protein interaction analysis, and other relevant experimental investigations.