After successful drug activation from signal-responsive prodrugs, we investigated their material compatibility to alkyne-substituted hydrogel scaffolds and drug release during signal activation by visual inspection of fluorescence intensity changes (Figure ). To assess concentration changes upon signal-triggered drug release, we used drug 3 (ofloxacin) as a model. Free ofloxacin exhibits strong blue emission under 365 nm UV irradiation, whereas its prodrug analogue emits green fluorescence (Supplementary Figure ), due to the quaternization of the piperazinyl motif on ofloxacin . This allows the visual distinction between prodrug 9 and free drug 3 under UV-light (Figure ).