The primary prerequisite for protein function is the attainment of an ordered three-dimensional structure enabled by proper protein folding. Often the goal of protein engineering is to increase both the soluble yield and functional efficacy of a binding protein or enzyme. However, in order to probe the function of analyte protein variants it is first necessary to isolate those that contain the desired folding properties. Particularly, it is desirable to perform these tasks in vivo to ensure relevance in the internal environment of cells. Using native cellular processes such as protein secretion, we have developed a set of genetic assays in bacteria that have enabled the laboratory evolution and isolation of folding-enhanced protein and peptide variants. We demonstrate the impact of these assays on antibody engineering, fluorescent protein evolution, and the isolation of difficult-to-fold recombinant proteins. These assays are high-throughput, robust, and should expand the toolbox available for researchers interested in fold-modifying proteins in laboratory evolution and protein engineering.