The broad inhibitory spectrum of aldehydes and the possibility that amino acid residues modulate their specificity point to the potential of using peptidyl aldehydes as activity-based probes. Here, we establish the potential of peptidyl aldehydes in activity-based proteomics by synthesizing different probes and using them to specifically label a well-known serine protease in an activity-dependent manner. From our results, peptidyl aldehydes emerge as promising activity-based probes that enable multiple enzymatic-class detection by substrate recognition and can be used in diverse activity-based proteomics applications like protein identification and activity profiling.