Activity-based proteomics is a methodology which is used to quantify the catalytically active sub-fraction of enzymes present in complex mixtures such as lysates or living cells. In order to apply this approach for in-cell selectivity profiling of inhibitors of serine proteases, we have designed a novel activity-based probe (ABP). This ABP consists of (i), a fluorophosphonate reactive group, directing the probe towards serine hydrolases or proteases and (ii), an alkyne functionality which can be specifically detected at a later stage with an azide-functionalized reporter group through a copper(I)-catalyzed coupling reaction ("click-chemistry"). This novel ABP was shown to label the active site of several serine proteases with greater efficiency than a previously reported fluorophosphonate probe. More importantly, our probe was cell-permeable and achieved labeling of enzymes within living cells with similar efficiency to that observed for the corresponding lysate fraction. Several endogenous serine hydrolases, whose activities were detected upon in-cell labeling, were identified by 2D gel and MS analyses. As a proof of principle, cell-permeable inhibitors of an endogenous serine protease (prolyl endopeptidase)were assessed for their potency and specificity in competing for the in-situ labeling of the selected enzyme. Altogether, these results open new perspectives for safety profiling studies in uncovering potential cellular "side-effects" of drugs (unanticipated off-target inhibition or activation) which may be overlooked by standard selectivity profiling methods.