Chemical probes that covalently modify the active sites of enzymes in complex proteomes are useful tools for identifying enzyme activities associated with discrete (patho) physiological states. Researchers in proteomics typically use two types of activity-based probes to fulfill complementary objectives: fluorescent probes for rapid and sensitive target detection and biotinylated probes for target purification and identification. Accordingly we hypothesized that a strategy in which the target detection and target isolation steps of activity-based proteomic experiments were merged might accelerate the characterization of differentially expressed protein activities. Here we report the synthesis and application of trifunctional chemical proteomic probes in which elements for both target detection (e.g. rhodamine) and isolation (e.g. biotin) are appended to a sulfonate ester reactive group, permitting the consolidated visualization and affinity purification of labeled proteins by a combination of in-gel fluorescence and avidin chromatography procedures. A trifunctional phenyl sulfonate probe was used to identify several technically challenging protein targets, including the integral membrane enzyme 3beta-hydroxysteroid dehydrogenase/Delta5-isomerase and the cofactor-dependent enzymes platelet-type phosphofructokinase and type II tissue transglutaminase. The latter two enzyme activities were significantly up-regulated in the invasive estrogen receptor-negative (ER(-)) human breast cancer cell line MDA-MB-231 relative to the non-invasive ER(+) breast cancer lines MCF7 and T-47D. Collectively these studies demonstrate that chemical proteomic probes incorporating elements for both target detection and target isolation fortify the important link between the visualization of differentially expressed enzyme activities and their subsequent molecular identification, thereby augmenting the information content achieved in activity-based profiling experiments.