Convenient, reliable detection of transmembrane protein topology, especially the orientation of the amino (N-) and carboxyl (C-) termini of a membrane-spanning segment, may aid in identifying protein-protein interactions and clarifying the important biological functions of proteins. Self-complementing split fluorescent proteins have been widely used to image protein-protein interactions, label endogenous proteins and visualize mRNA localization. Here, we expand this toolset and develop an efficient method combining a self-complementing split mNeonGreen2 with site-directed labeling (SSDL) to identify the topology of transmembrane proteins. With SSDL, for the first time, we clearly demonstrate that both the N- and C-termini of etoposide-induced protein 2.4, which localizes in the endoplasmic reticulum, have a cytosolic orientation. This method can be useful for determining the topology of other organelle-based transmembrane proteins that have insufficient structural information.