Matriptase-2 is a type II transmembrane serine protease and the key regulator of systemic iron homeostasis. After its synthesis, matriptase-2 is present on the cell surface as an inactive zymogen which is activated by processing. Two processing steps lead to the release of a 55 kDa enzymatically active fragment of the protein into the cell culture supernatant. Since the underlying mechanism of these processing steps is not yet fully understood there is strong need for analytical tools that help to distinguish active and inactive matriptase-2. For this purpose, we present a short biotinylated peptide probe with a chloromethyl ketone group as an inhibitor and activity-based probe for matriptase-2. This probe was characterized in kinetic experiments and applied for in-gel detection of matriptase-2.