Target-based discovery of first-in-class therapeutics demands an in-depth understanding of the molecular mechanisms underlying human diseases. Precise measurements of cellular and biochemical activities are critical to gain mechanistic knowledge of biomolecules and their altered function in disease conditions. Such measurements enable the development of intervention strategies for preventing or treating diseases by modulation of desired molecular processes. Fluorescence-based techniques are routinely employed for accurate and robust measurements of activity of molecular targets and for discovering novel chemical molecules that modulate the activity of molecular targets. In the current review, the authors focus on the applications of fluorescence-based high throughput screening (HTS) and fragment-based ligand discovery (FBLD) techniques such as fluorescence polarization (FP), Förster resonance energy transfer (FRET), fluorescence thermal shift assay (FTSA) and microscale thermophoresis (MST) for the discovery of chemical probe to exploring target’s role in disease biology and ultimately, serve as a foundation for drug discovery. Some recent advancements in these techniques for compound library screening against important classes of drug targets, such as G-protein-coupled receptors (GPCRs) and GTPases, as well as phosphorylation- and acetylation-mediated protein-protein interactions, are discussed. Overall, this review presents a landscape of how these techniques paved the way for the discovery of small-molecule modulators and biologics against these targets for therapeutic benefits.