Activation kinetics of metallo-prodrugs control the types of possible interactions with biomolecules. The intact metallo-prodrug is able to engage with potential targets by purely non-covalent bonding, while the activated metallodrug can form additional coordination bonds. It is hypothesized that the additional coordinative bonding might be favourable with respect to the target selectivity of activated metallodrugs. Thus, a time-dependent shotgun proteomics study was conducted in HCT116 colon carcinoma cells with plecstatins, which are organoruthenium anticancer drug candidates. First, the target selectivity was evaluated in a time-dependent fashion, which accounted for their hydrolysis kinetics. The binding selectivity increased from 50- to 160-fold and the average specificity from 0.72 to 0.86, respectively, from the 2 h to the 4 h target profiling experiment. Target profiling after 19 h did not reveal significant enrichments, possibly due to deactivation of the probe via arene cleavage. Up to 450 interactors were identified in the target profiling experiments. A plecstatin analogue that substituted a hydrogen bond acceptor with a hydrogen bond donor abrogated the target selectivity for plectin in HCT116 whole cell lysates, underlining the necessity of this hydrogen bond acceptor for a strong interaction between plecstatin and plectin. Second, time-dependent response profiling experiments provided evidence that plecstatin-2 induced an integrated stress response (ISR) in HCT116 cell culture. The phosphorylation of eIF2α, a key mediator of the ISR, after 3 h treatment indicated that this perturbation was initiated by the intact plecstatin-2 prodrug, while the effects of plectin-targeting are mediated by activated plecstatin-2.