Translocation of the apoptosis-inducing factor (AIF) from the mitochondria to the nucleus is crucial for AIF-mediated apoptosis. However, the lack of methods for real-time spatial and temporal analysis of translocation of functional AIF is a large hurdle to gain a detailed understanding of this process. In this study, a genetic code expansion technique was developed to overcome this hurdle. Specifically, this technique was utilized to construct ANAP-AIF containing a small fluorescent amino acid (ANAP) at a specific site in cells. Additionally, we developed efficient fluorescence resonance energy-transfer systems consisting of ANAP-AIF and either yellow fluorescent protein (YFP)-fused cyclophilin A (CypA) or Hsp70, respective positive and negative regulators for AIF translocation to the nucleus. We found that apoptosis inducers, including apoptozole, 2-phenylethynesulfonamide (PES), myricetin, Bam7, reactivating p53 and inducing tumor apoptosis (RITA), brefeldin A, and carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) promote translocation of mitochondrial AIF to the cytosol after 4 h incubation, reaching a maximum after 6-7 h. However, these substances did not enhance AIF translocation to the nucleus through the interaction of AIF with Hsp70 in the cytosol. On the other hand, treatment with apoptosis inducers, such as paclitaxel, silibinin, doxorubicin, actinomycin D, and camptothecin caused AIF translocation to the nucleus after 4 h incubation through AIF binding to CypA, reaching saturation after 6-7 h. It was also found that Hsp70 and CypA regulate AIF translocation in a mutually exclusive manner because they do not interact with AIF simultaneously in cells undergoing apoptosis. The results demonstrate clearly that ANAP-incorporated proteins are powerful to obtain a more in-depth understanding of protein translocation.