Laundry detergent formulations include proteases to digest and clean protein-based stains. Methods that can profile protease activity and specificity in detergents could be important in guiding the development of engineered proteases for these applications. The work reported here uses peptide arrays with analysis by self-assembled monolayers for matrix-assisted laser desorption/ionization (SAMDI) mass spectrometry to analyze protease activity across a 324-peptide library for five commercial laundry detergents. The results showed differences in cleavage activity across different brands. The combination of peptide arrays and SAMDI mass spectrometry provides a rapid and reliable technique for analyzing protease activities in laundry detergents and has the potential to play an important role in the development of new proteases and detergent formulations.