This lecture covers the design optimization of Snif-it's sensors, focusing on geometry, sequence of domains, spacing, and circular permutation, aiming to understand the molecular basis for changes in FRET ratio and the effective concentration of intra-molecular ligands. It also introduces semi-synthetic sensors for visualizing metabolite concentrations and LUCID's bioluminescent sensor proteins for drug monitoring, including the principles behind them and their applications in point-of-care therapeutic drug monitoring.