A new method for evaluating the substrate efficiency of deoxynucleoside triphosphates containing functional groups for the selection of modified aptamers (mod-SELEX) is proposed. The method involves conducting three consecutive rounds of PCR with a combinatorial library and a modified dNTP candidate for mod-SELEX. The conclusion about the applicability of a specific dNTP derivative is made by the nature of the change in the amplification curve during the three rounds of PCR in real time and does not require SELEX rounds. If the library degenerates during amplification (becomes less representative), it means that the specific modification of dNTP cannot be used with the selected polymerase and the other selected library amplification conditions, since it leads to competitive amplification. When the nature of the signal accumulation curve does not change, it is concluded that the modified triphosphate does not affect the distribution of oligonucleotides with different sequences in the library, that is, it does not lead to a change in its composition from the point of view of the applied detection method. It is these derivatives that can be applied with the selected conditions for the selection of aptamers. The method is applicable for quick assessment of the substrate suitability of modifications introduced into deoxynucleoside triphosphates for mod-SELEX and will be useful in the selection of aptamers for clinical diagnostics, medicine and scientific research.