Staphylococcus aureus is a dangerous human pathogen characterized by growing antibiotic resistance. Virulence of S. aureus relies on a variety of secreted and cell surface associated virulence factors among which certain proteolytic enzymes play an important role. Amid staphylococcal extracellular proteases, those encoded by the spI operon remain poorly characterized, both in terms of enzymology and their physiological role. Initial data demonstrated that SpI proteases exhibit restricted substrate specificity. This study describes development of convenient protein FRET substrates for SpIB protease and characterization of the substrate preference of the protease at the P1' position. Kinetic data on hydrolysis of a panel of substrates substituted at the said position is provided.