A ligation strategy based on the Diels-Alder [4+2] cycloaddition for the two-step activity-based labeling of endogenously expressed enzymes in complex biological samples has been developed. A panel of four diene-derivatized proteasome probes was synthesized, along with a dienophile-functionalized BODIPY(TMR) tag. These probes were applied in a Diels-Alder labeling procedure that enabled us to label active proteasome beta-subunits selectively in cellular extracts and in living cells. We were also able to label the activity of cysteine proteases in cell extracts by utilizing a diene-derivatized cathepsin probe. Importantly, the Diels-Alder strategy described here is fully orthogonal with respect to the Staudinger-Bertozzi ligation, as demonstrated by the independent labeling of different proteolytic activities by the two methods in a single experiment.