Abstract: The fusing of a protein of interest to a fluorescent protein followed by fluorescence microscopy is a very common method of determining protein localization and dynamics. However even small fluorescent proteins can be large enough to affect protein folding and localization, therefore the ability to use a smaller tag but still be able to detect a fluorescent signal in live cell imaging experiments is extremely valuable. Theself-assembling split sfGFPOPT system allows the fusion of the protein of interest with the11thβ-strand of super-folderGFP(sfGFP11) which is only 13 amino acids long. When this construct is delivered into protoplasts made from transgenic plants expressing sfGFP1-10 (sfGFP1-10OPT) targeted to the desired compartment, the two parts assemble and fluorescence is reconstituted that can be detected by confocal laser scanning microscopy. Here, we present the application of this method for protein targeting to plant peroxisomes using Catalase (CAT2 of Arabidopsis thaliana) as an example. As peroxisomes are able to import folded and oligomeric proteins, careful consideration of appropriate controls is also required to ensure correct interpretation of the results.