Genome sequencing projects have uncovered many novel enzymes and enzyme classes for which knowledge of active site structure and mechanism is limited. To facilitate mechanistic investigations of the numerous enzymes encoded by prokaryotic and eukaryotic genomes, new methods are needed to analyze enzyme function in samples of high biocomplexity. Here, we describe a general strategy for profiling enzyme active sites in whole proteomes that utilizes activity-based chemical probes coupled with a gel-free analysis platform. We apply this gel-free strategy to identify the sites of labeling on enzymes targeted by sulfonate ester probes. For each enzyme examined, probe labeling was found to occur on a conserved active site residue, including catalytic nucleophiles (e.g., C32 in glutathione S-transferase omega) and bases/acids (e.g., E269 in aldehyde dehydrogenase-1; D204 in enoyl CoA hydratase-1), as well as residues of unknown function (e.g., D127 in 3 beta-hydroxysteroid dehydrogenase/isomerase-1). These results reveal that sulfonate ester probes are remarkably versatile activity-based profiling reagents capable of labeling a diversity of catalytic residues in a range of mechanistically distinct enzymes. More generally, the gel-free strategy described herein, by consolidating into a single step the identification of both protein targets of activity-based probes and the specific residues labeled by these reagents, provides a novel platform in which the proteomic comparison of enzymes can be accomplished in unison with a mechanistic analysis of their active sites.