The investigation of cellular processes on the molecular level is important to understand the functional network within plant cells. self-assembling GFP has evolved to be a versatile tool for (membrane) protein analyses. Based on the autocatalytical reassembling property of the nonfluorescent strands 1–10 and 11, protein distribution and membrane protein topology can be analyzed in vivo. Here, we provide basic protocols to determine membrane protein topology in Arabidopsis thaliana protoplasts.