We describe detailed procedures for the chemical synthesis of two costly chromogenic substrates, H-D-Ile-L-Pro-L-Arg-p-nitroanilide (S2288) and H-D-Val-L-Leu-L-Lys-p-nitroanilide (S2251), which are widely employed for assay of tissue plasminogen activator and plasmin, respectively, as well as in inhibitor assays for these enzymes. Quantities of 5 grams or more of these reagents can be synthesized on a bench-top scale with routine equipment in a laboratory or facility with minimal experience in peptide chemistry. While some of the chemical reactions have been employed previously, improvements have been made therein in this manuscript, and these detailed descriptions are mainly provided to be of service to investigators who employ large quantities of these substrates in their work. The main strategic difference between our methodology and that employed previously is the coupling of the L-Lys- and L-Arg-p-nitroanilides to the previously synthesized, and otherwise completed peptides, which gives superior yields of final product and provides flexibility the chemical synthesis of substrates with other amino acids present.