Specific bacteria imaging is highly desirable in clinical diagnostics. Probes enabling rapid and specific diagnostics of bacteria are limited. Current clinical infection diagnostics is time consuming and invasive, relies on microbiological cultures. We investigated the potential of Lysostaphin as a specific probe to label staphylococci in a new labeling protocol. We used azido (N(3))-modified Lysostaphin-N(3) and DIBO-dye in a two-step bacteria-labeling protocol. N(3) and DIBO (di-benzocyclooctyne) are the counterparts of the "click" chemistry. In the first step, Lysostaphin-N(3) binds specifically to Staphylococcus aureus. In the second step, N(3) clicks to DIBO thus achieving the selective for S. aureus labeling. Such a two-step approach effectively distinguishes S. aureus from Escherichia coli; non-toxic and was proven to work in vivo. The two-step labeling protocol is a promising approach for diagnostic imaging of staphylococcal infections in clinical settings.