As the SERS spectrum of Cys-cabn and Cys-cabn/Cys-CABN mixtures adsorbed onto AgNPs was found to depend on the concentration of cysteine aliquots added to the AgNPs colloidal suspensions, the optimization of the cysteine concentration was necessary prior to further experimentation. SERS spectra were collected for samples containing various concentrations of cysteine aliquots (0.25, 0.50, and 1.00 mM), both for the simultaneous coadsorption of Cys-cabn and Cys-CABN (20 µL of equimolar mixture), as well as the two-step adsorption of Cys-cabn followed by addition of Cys-CABN after a 10-minute delay (10 µL aliquots). The comparison of SERS results for such prepared samples is presented in Fig. . The lowest concentration tested (0.25 mM) resulted in both SERS marker bands presenting similar intensity for the two-step adsorption experiment, suggesting lack of competition in binding and, therefore, unsaturated surface coverage. Furthermore, the signal-to-noise ratio of the SERS spectrum was rather unsatisfactory. On the other hand, the concentration of 1 mM resulted in the band arising from Cys-CABN (the second isotopologue added), to be of relatively low intensity compared to Cys-cabn. Thus, the concentration of 0.5 mM was chosen for further experiments, as it displayed a sufficient relative intensity of marker bands in the two-step adsorption experiment, allowing for a more accurate SERS analysis.