Two-component systems (TCSs) are the largest class of biological signal transduction pathways and an important class of bacterial sensors. In their native contexts, TCSs enable bacteria to sense and respond to changes in their environment. TCSs are also a major source of novel biosensors for medical, environmental, and industrial applications. In the first portion of this work, we use TCS engineering and chemical input screens to characterize TCSs of unknown function from Shewanella oneidensis. Through these screens, we discover a pH-responsive TCS, which we apply to detect acidification of intestinal tissues in a mouse model of inflammatory bowel disease. In the second portion of this work, we develop a high-throughput screening approach for characterizing peptide-TCS interactions. We screen PhoPQ, a virulence-regulating TCS from Salmonella Typhimurium, against 117 human antimicrobial peptides (AMPs). We discover 13 novel activators of PhoPQ comprising diverse sequences, structures, and biological functions and identify subdomains and peptide biophysical features responsible for PhoPQ activation. Finally, we find that PhoPQ homologs exhibit distinct AMP response profiles, suggesting a role for evolutionary adaptation in AMP sensing. The engineering approaches developed here enable high-throughput discovery and characterization of TCS inputs, which could provide important insights into bacterial stimulus response and reveal novel biosensors with applications across a wide range of sectors.