Nowadays, the analysis of the uptake and intracellular distribution of cell-penetrating peptides mostly relies on fluorescence microscopy, using fluorescently labeled CPP analogs. However, fluorescence microscopy does not reveal to which degree fluorescence reflects the intact peptide or only breakdown products. Here, we introduce fluorescence correlation spectroscopy (FCS) as a powerful method to address peptide stability in cells and cell lysates. Measurements in lysates of cells incubated with peptide yield information on degradation of the total cellular peptide content. In combination with protease inhibitors, such measurements enable conclusions on trafficking pathways. Intracellular FCS measurements provide direct information on peptide degradation and association with cellular structures in intact cells.