Understanding the cell biology of many proteins requires knowledge of their in vivo topological distribution. Here we describe a new fluorescence-based technique, fluorescence protease protection (FPP), for investigating the topology of proteins and for localizing protein subpopulations within the complex environment of the living cell. In the FPP assay, adapted from biochemical protease protection assays, GFP fusion proteins are used as noninvasive tools to obtain details of protein topology and localization within living cells in a rapid and straightforward manner. To demonstrate the broad applicability of FPP, we used the technique to define the topology of proteins localized to a wide range of organelles including the endoplasmic reticulum (ER), Golgi apparatus, mitochondria, peroxisomes and autophagosomes. The success of the FPP assay in characterizing the topology of the tested proteins within their appropriate compartments suggests this technique has wide applicability in studying protein topology and localization within the cell.