Type III protein secretion is essential for many Gram-negative bacterial infections of host cells and an attractive target for new antibacterial drugs. Here, we describe a bacterial protein effector-carboxypeptidase G2 (CPG2) reporter system for fluorescence and visible detection of type III protein secretion in Salmonella typhimurium. This system provides a general method for measuring protein expression and secretion as well as a high-throughput and quantitative assay for analyzing type III protein secretion inhibitors.