Luminescent imaging has become a pivotal technique for monitoring enzymatic activity, offering a high signal-to-noise ratio by avoiding external light sources. Traditional methods of coupling aminoluciferin to peptides have faced challenges due to the instability of the aminoluciferin. We have developed a solid-phase peptide synthesis (SPPS) protocol involving anchoring of P1 lysine or arginine residues to a solid support, followed by subsequent elongation and aminoluciferin incorporation post-resin cleavage. An optimized benzotriazole-based guanidinylation reagent was used for efficient large-scale synthesis with minimal byproducts. This method enhances the development of substrates for trypsin-like serine proteases by facilitating the synthesis of novel luminescent activity-based probes.