Reliable enzymatic digestion is crucial for successful mass spectrometry-based proteomics. This study compares the arginine-specific protease, GingisREX, with a traditional trypsin/lys-C mixture for identifying E. coli proteins. Using GingisREX resulted in more protein identifications due to fewer peptides per protein, creating a simpler mixture. GingisREX also showed better digestion efficiency, fewer missed cleavages, and improved MS/MS data quality for larger peptides. These findings suggest GingisREX is a valuable complement to trypsin for detecting bacterial proteins and could be optimized as an effective alternative for identifying HCPs in biotherapeutics.