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Matriptase-2 is a type II transmembrane serine protease and a key regulator of systemic iron homeostasis. Since the activation mechanism and several features of the physiological role of matriptase-2 are not fully understood, there is strong need for analytical tools to perform tasks such as distinguishing active and inactive matriptase-2. For this purpose we present a short biotinylated peptide derivative with a chloromethyl ketone group, biotin-RQRR-CMK, as an activity-based probe for matriptase-2. Biotin-RQRR-CMK was kinetically characterized and exhibited a second-order rate constant of inactivation (kinac/Ki) of 10,800 M−1 s−1 towards the matriptase-2 activity in the supernatant of transfected human embryonic kidney (HEK) cells. Biotin-RQRR-CMK was able to label active matriptase-2, as visualized in western blot experiments. Pretreatment with aprotinin, an active-site directed inhibitor of serine proteases, protected matriptase-2 from the reaction with biotin-RQRR-CMK.