There is provided a method for determining the activity of a protease in a sample. The method comprises (i) admixing said sample with a substrate, wherein the substrate has the formula (1a) wherein: R1 is a hydrocarbyl group; R2 is a first peptide moiety; R3 is a second peptide moiety and X is selected from the group consisting of O, S and NH; Y1 is a suitable substituent; Y2 is a suitable substituent; and (ii) determining the activity of said protease by detecting the presence of a reporter having the formula H—X—R1, wherein: X is selected from the group consisting of O, S and NH; R1 is a hydrocarbyl group. The substrate and reporter are useful for determining the efficacy of protease-modulators and candidate protease-modulators and in the diagnosis of a disease or disorder in a subject.