The trimethylsilyl (TMS) group in the amino acid N6-(((trimethylsilyl)-methoxy)carbonyl)-L-lysine (TMSK) shows an intense narrow signal near 0 ppm in 1D 1H-NMR spectra, which can easily be identified even for high molecular weight systems without the need for isotopic labelling.1 Similarly, the amino acid N6-trifluoroacetyl-L-lysine (TFAK) allows selective detection by 19F-NMR spectroscopy with good sensitivity.2 Both amino acids can be incorporated into proteins in response to an amber-stop codon.1,2 The present work3 is a benchmark study of these NMR probes for the site-specific detection of ligand binding. TMSK or TFAK were installed in the 70 kDa SARS-CoV-2 main protease (Mpro) homodimer at solvent-exposed sites either close or not close to the binding site to interrogate the binding of previously identified compounds.4,5 In titration experiments conducted with increasing concentrations of inhibitors, the NMR signals of TMSK or TFAK installed at different sites responded in fundamentally different ways depending on the proximity of the TMSK or TFAK residue to the bound inhibitor. When located near a bound inhibitor, both probes displayed either new signals, significant line-broadening or line-narrowing effects. The spectral changes were much less pronounced when the probes were installed in locations remote from the ligand binding site. Interestingly, neither TMSK nor TFAK confirmed the binding site of the compound pelitinib, which had previously been identified as an allosteric inhibitor of Mpro.5 In fact, subsequent activity assays revealed that pelitinib acts as an enhancer rather than an inhibitor of Mpro activity by promoting dimerization.3 These results show that TMSK and TFAK are useful NMR probes for difficult proteins. In the case of Mpro, the chemical shift changes of backbone 15N-1H groups affected by the binding of the inhibitor delivered much more ambiguous results,4 which could be attributed to allosteric responses.