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Summary: Here, we describe a protocol for the translational regulation of transfected messenger RNAs (mRNAs) using light in mammalian cells. We detail the steps for photocaged ligand synthesis, template DNA preparation, and mRNA synthesis. We describe steps for mRNA transfection, treatment of cells with a photocaged ligand followed by light irradiation, and analysis of the transgene expression. The protocol enables spatiotemporally regulated transgene expression without the risk of insertional mutagenesis.For complete details on the use and execution of this protocol, please refer to Nakanishi et al. (2021). : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.