based kinetic monitoring in the fluorination of bioactive compounds serves many benefits in the drug discovery process, since fluorinated motifs additionally improve drug pharmacology. In this study, 19 F NMR spectroscopy was utilized to monitor the synthesis of novel trifluorinated analogs of monastrol, a small molecule dihydropyrimidinone kinesin-Eg5 inhibitor, and to probe the mechanism of the Biginelli cyclocondensation, a multicomponent reaction used to synthesize dihydropyrimidinone and tetrahydropyrimidinones through a Bronsted-or Lewis-acid catalyzed cyclocondensation between ethyl acetoacetate, thiourea, and an aryl aldehyde. In the present study, a trifluorinated ketoester serves a dual purpose as being the source of the trifluoromethyl group in our fluorinated dihydropyrimidinones and as a spectroscopic handle for real time reaction monitoring and tracking of reactive intermediates by 19 F NMR. Further, upon extending this workflow to a diverse array 3-and 4-substituted aryl aldehydes, we were able to derive Hammett linear free energy relationships (LFER) to determine stereoelectronic effects of paraand meta-substituted aryl aldehydes to corresponding reaction rates and mechanistic routes. In addition, we used density functional theory (DFT) calculations to corroborate our experimental results through the thermodynamic values of key intermediates in each mechanism. Finally, these studies culminate in the synthesis of a novel trifluorinated analog of monastrol and its subsequent biological evaluation in vitro. More broadly, we show an application of benchtop 19 F NMR spectroscopy as an analytical tool in the real-time investigation of a mechanistically and chemically complex multicomponent reaction mixture.