Releasing bioactive molecules in specific subcellular locations from the corresponding caged precursors offers great potential in photopharmacology, especially when using biologically compatible visible light. By taking advantage of the intrinsic preference of COUPY coumarins for mitochondria and their long wavelength absorption in the visible region, we have synthesized and fully characterized a series of COUPY-caged model compounds to investigate how the structure of the coumarin caging group affects the rate and efficiency of the photolysis process. Uncaging studies using yellow (560 nm) and red light (620 nm) in phosphate-buffered saline medium have demonstrated that the incorporation of a methyl group in a position adjacent to the photocleavable bond is particularly important to fine-tune the photochemical properties of the caging group. Additionally, the use of a COUPY-caged version of the protonophore 2,4-dinitrophenol allowed us to confirm by confocal microscopy that photoactivation can occur within mitochondria of living HeLa cells upon irradiation with low doses of yellow light. The new photolabile protecting groups presented here complement the photochemical toolbox in therapeutic applications since they will facilitate the delivery of photocages of biologically active compounds into mitochondria.