Phytochrome A (phyA) is the far-red (FR) light photoreceptor in plants, and its phosphorylation status plays a critical role in modulating its protein activity and function. The protoplast transient expression system has become an increasingly efficient approach for analyzing promoter activities, protein subcellular localization, protein–protein interactions, etc. Recently, we employed the protoplast transient expression system to assess the role of the protein kinases PHOTOREGULATORY PROTEIN KINASES (PPKs) in phosphorylating phyA. TANDEM ZINC-FINGER/PLUS3 (TZP), a phyA-interacting protein previously shown to mediate phyA phosphorylation in vivo, was coexpressed with phyA and PPKs in Arabidopsis protoplasts. After the treatment with FR light, we observed that PPKs can directly phosphorylate phyA and that TZP enhances PPKs-mediated phosphorylation of phyA in Arabidopsis protoplasts. In this chapter, we will describe the detailed experimental procedures and the key points for implementing this system. This system may also be feasible for validating the relationship between other kinases and their potential substrates.