The glutathione-mediated retro Michael-type addition reaction is demonstrated to take place at the interface of small water-soluble maleimide-functionalized gold nanoparticles (Maleimide-AuNP). The retro Michael-type addition reaction can be blocked by hydrolyzing the Michael addition thioether adduct at the nanoparticle's interface under reaction conditions that do not cause AuNP decomposition. This procedure "locks" the molecule of interest onto the Maleimide-AuNP template for potential uses in medical imaging and bioconjugation, ensuring no loss of the molecular cargo from the nanocarrier. On the other hand, the glutathione-mediated retro Michael-type addition reaction can be exploited for delivering a molecular payload. As a proof of concept, a fluorogenic molecular cargo was incorporated onto a Maleimide-AuNP and delivered via the glutathione-mediated retro Michael-type addition reaction.