mRNA-based transfection is an attractive strategy for manipulation of gene expression for gain-of-function studies and therapeutic applications. As a potential therapeutic regulator, mRNA transfection has mainly been hampered by poor delivery strategies, combined with lack of specific targeting to the intended tissue(s) or cells. In this chapter, we describe a protocol for light-induced mRNA transfection into human cancer cell lines with the benefit for time- and site-specific mRNA targeting. Light-induced mRNA transfection is achieved by delivering mRNA molecules into endosomal and lysosomal vesicles. Subsequently, a photosensitizer (PS) localized in the membranes of these vesicles is used to induce damage, resulting in release of mRNA molecules into the cytosol. The main benefit of the strategy proposed is the possibility for protein production from the delivered mRNA in a way that is controllable in a time- and site-specific manner.