Numerous Gram-negative bacteria utilize the twin-arginine translocation (Tat) pathway for the export of fully-folded proteins from the cytoplasm and into the periplasmic space. Using a mini-Tn5 transposon system, we have engineered an alkaline phosphotase (PhoA)-based screen for genome-wide detection of Tat transport. Additionally, by toggling the redox state of a specially-engineered strain of Escherichia coli, we are able to discriminate between protein translocation via the Tat pathway and its well-studied counterpart, the general secretory (Sec) pathway. We also demonstrate the ability of this system to serve as a surrogate for robust detection of Tat substrates that are not native to E. coli. Coupled with in silico predictions of secreted proteins, this screen allows for rapid experimental confirmation of predicted substrates and for discovery of non-canonical substrates that evade bioinformatics. The significance of these results will be discussed in the context of bacterial pathogenesis and also biotechnology applications that hinge on protein secretion.