Existing monitoring methods for protein phosphorylation involved in intracellular signal transduction in vivo are exclusively based on fluorescence resonance energy transfer, which needs the measurement of the change in fluorescence intensities at two wavelengths. Therefore, it is difficult to monitor protein phosphorylation together with other related signaling processes, such as second messengers and protein translocation. To overcome this problem, we developed novel fluorescent indicators, each containing a differently colored (cyan and green) single fluorophore. The present indicator is a tandem fusion protein containing a kinase substrate domain, a circularly permuted fluorescent protein (cpFP), and a phosphorylation recognition domain. The cpFP is obtained by dividing a green fluorescent protein mutant (GFP) at residue 144-145 and linking the carboxy and amino portions thereof with a peptide linker. The substrate domain used in this study is a peptide sequence that is phosphorylated by insulin receptor. Phosphorylation of the substrate domain induces its interaction with the phosphorylation recognition domain, which causes a conformational change in the cpFP and a change in its fluorescence. The cyan and green indicators exhibited 10% decrease and 15% increase, respectively, in their fluorescence intensities upon phosphorylation. Using this cyan indicator and GFP-tagged mitogen-activated protein kinase (MAPK), we found that insulin-induced protein phosphorylation occurred immediately upon the addition of insulin, whereas nuclear translocation of MAPK occurred 7 min later. By tailoring the substrate domains and the phosphorylation recognition domains in these cyan and green indicators, the present approach should be applicable to the in vivo analysis of a broad range of protein phosphorylation processes, together with other intracellular signaling processes.