The importance of fluorine in medicinal chemistry is well‐recognized. An increasing number of drugs on the market contain fluorine, the presence of which is often of major importance to activity [ 1-4]. The special nature of fluorine imparts a variety of properties to certain molecules, including increased permeability, favorable protein–ligand interactions, metabolic stability, changes in physical properties, and selective reactivities [ 5-12]. More recently, fluorine NMR spectroscopy, a somewhat neglected technique in the bio‐NMR community (for a comprehensive review of math NMR applied to proteins, see Gerig [ 13]), has emerged as a powerful tool in drug discovery. Several industrial and academic groups have recognized its potential and are now investing in this methodology. The technique is used in the hit identification and validation phase, and in the hit‐to‐lead phase of drug discovery projects. Another useful application of the technique that is outside the scope of this chapter is in the in vivo and in vitro characterization of fluorinated drugs (for reviews, see, e.g., Bachert [ 14] and Malet‐Martino et al. [ 15]). The renewed interest in math NMR spectroscopy has been made possible by technical developments that have resulted in an improvement in sensitivity of the detection of fluorine signals [ 16], and by convincing experimental and theoretical demonstrations of the relative sensitivity of the methodology for detecting weak interactions between small molecules and biological targets where other biophysical techniques fail [ 17-19]. Fluorine NMR spectroscopy can be used for performing binding and biochemical assays directed at the identification of enzyme and/or protein–protein, protein–DNA, and protein–RNA interaction inhibitors [ 20-22]. The binding assays are carried out in a direct‐mode format by screening mixtures of fluorinated molecules [ 18, 23-34] or in a competition‐mode format in the presence of a low‐affinity fluorinated spy molecule [ 23, 24, 30, 33-36]. The theoretical and technical aspects of these binding assays have been treated in depth in recent publications and review articles [ 17, 20]. These experiments, in particular those that measure a reduction in the math transverse relaxation T 2 in the presence of the biological target, are among the most sensitive binding assays for the identification of very weak affinity ligands due to the large dynamic range [ 19]. This originates from the large math chemical shift anisotropy and chemical exchange contributions to the transverse relaxation of the math signals in the presence of the biological target. In this chapter we focus on the application of math NMR spectroscopy to simple and complex biochemical assays for the detection of inhibitors or agonists of enzymatic reactions. The chapter is organized in several sections. An initial section introduces basic notions of enzymology required for the interpretation of the NMR results. The principles at the basis of the methodology are then presented and analyzed. Application of the technique starts with the simplest biological system of one substrate and one enzyme, followed by more complex biological systems, such as several substrates in the presence of one or more enzymes, which are relevant in systems biology. A section comparing the NMR methodology with other more established biophysical techniques often used for biochemical screening is then presented. Finally, protocols for performing the assays and for measuring the inhibition strength of the identified NMR hits along with NMR procedures for the identification of false positives are presented.