New approaches for molecular analysis continuously open new vistas in molecular medicine. Our lab has built a series of molecular detection techniques based on enzymatic ligation of synthetic oligonucleotides as versatile tools to gain new molecular insights. These fundamental techniques continue to yield new means for specific, high-throughput analyses of nucleic acids and proteins in contexts of relevance for molecular medicine. The combined potential for vast multiplexing and low sample consumption renders the assays described herein attractive as a basis for AI-assisted model building in medicine. Accordingly, this overview is aimed to present a ligase-based molecular toolbox to choose from in addressing present and upcoming analytical needs.