Here we present a platform for discovery of protease-activated prodrugs and apply it to antibiotics that target Gram-negative bacteria. Because cleavable linkers for prodrugs had not been developed for bacterial proteases, we used substrate phage to discover substrates for proteases found in the bacterial periplasm. Rather than focusing on a single protease, we used a periplasmic extract of E. coli to find sequences with the greatest susceptibility to the endogenous mixture of periplasmic proteases. Using a fluorescence assay, candidate sequences were evaluated to identify substrates that release native amine-containing payloads. We next designed conjugates consisting of (1) an N-terminal siderophore to facilitate uptake, (2) a protease-cleavable linker, and (3) an amine-containing antibiotic. Using this strategy, we converted daptomycin-which by itself is active only against Gram-positive bacteria-into an antibiotic capable of targeting Gram-negative Acinetobacter species. We similarly demonstrated siderophore-facilitated delivery of oxazolidinone and macrolide antibiotics into a number of Gram-negative species. These results illustrate this platform's utility for development of protease-activated prodrugs, including Trojan horse antibiotics.