Copper-catalyzed azide-alkyne cycloaddition (CuAAC) has been widely used in a variety of scientific research, including dynamic proteomics. The current well-established protocols of CuAAC for proteomics analysis introduce labeling tags (azide- or alkyne-containing reagents) at the protein level, followed by downstream analysis by mass spectrometry. In the present study, a new method for proteomic profiling of nascent proteins relying on highly efficient peptide-based click chemistry is proposed, in which the CuAAC reaction was performed at the peptide level, leading to a significant increase in efficiency of the click conjugation reaction. A remarkable improvement in identification rate for spectrum, distinct peptide, and protein was achieved when proteins to be analyzed were proteolytically cleaved into peptides prior to the click conjugation reaction, which would be beneficial to downstream proteomics analysis, especially for the detection of AHA-tagged proteins in very low amounts.