Protein-protein interactions (PPIs) are crucial for many diverse cellular processes, and dysregulated PPIs are often implicated in disease states. Therefore, monitoring PPIs is critical to understanding underlying biological processes and disease. Many different techniques and tools have been developed to monitor PPIs, including biosensors. Biosensors, which are composed of biological components, contain detection and response elements to facilitate transducing a biochemical signal or event into a detectable output. Certain biosensors with the ability to generate genetic outputs or protein outputs have been utilized for monitoring PPIs and synthetic biology applications including, the generation of synthetic genetic circuits and biosynthetic pathways, diagnostic tools and therapeutics, and sensors for industrial applications. However, the extensive, system-specific engineering and optimization required for many of these biosensors precludes their use in other biosensor designs and applications. Because of the utility of biosensors to sense, monitor, and impact cellular biology, there is a growing demand in many diverse fields for biosensors that are highly characterized and broadly applicable. My thesis work aimed to generate such a biosensor for PPIs; a sensor that was robust, versatile, and capable of being implemented in orthogonal systems and contexts without the need for extensive re-optimization for each new PPI. By utilizing the T7 RNA polymerase (RNAP) as a scaffold, we developed a new protein fragment complementation assay (PCA)-based biosensor for the detection of PPIs. To optimize the properties of the split T7 RNAP biosensor scaffold, we utilized the directed evolution platform phage assisted continuous evolution (PACE) with a dual positive and negative selection scheme. The resultant split T7 RNAP PCA biosensor scaffold was characterized with multiple PPIs to show its versatility, including light-inducible and small molecule-inducible PPIs; and its orthogonality was demonstrated by testing in both E. coli and mammalian cells. The applicability of the split T7 RNAP biosensor scaffold for novel functions was demonstrated by generating a selection based scheme to interrogate the PPI interface of the KRAS/RAF PPI. Without the need to optimize any component of the split T7 RNAP system, a selection of different RAF variants against KRAS was conducted in order to identify key residues in the interaction interface. Preliminary work with utilizing the split T7 RNAP biosensor scaffold for directed evolution applications was also explored by using stable protein scaffolds, termed antibody mimetics, as starting points to evolve a new PPI partner for the inflammatory bowel disease (IBD) biomarker calprotectin. From the original characterization and these additional applications, we were able to demonstrate how the split T7 RNAP biosensor is a useful tool that can be applied to diverse applications, and should find broad utility in synthetic biology applications.