Proteomics is a fast-growing discipline that aims at systematic identification, quantification of proteins and their post-translational modifications in cells. Mass spectrometry-based shotgun proteomics technology is currently one of the mainstream methods for proteomics research. With this method, proteins need to be digested to peptides by site-specific proteases before they can be detected with mass spectrometry. Therefore, site-specific proteases played key roles in this process and so far, a variety of specific proteases have been developed and used in proteomics study. Particularly, the identification, characterization and development of proteases that cleave at the N-termini of corresponding amino acid residues, which are just mirrors to those of typical C-termini proteases, provide novel tools for proteomics analysis. In this review, we summarized the proprieties of LysargiNase, a most recently identified mirror trypsin, and its applications in proteomics research to promote its more widespread usage.