Cell‐penetrating peptides are known to penetrate cells through endocytosis and translocation. The two pathways are hardly distinguished in current cell assays. We developed a reliable, simple and robust method to distinguish and quantify independently the two routes. The assay requires (DABCYL) 4‐(dimethylaminoazo)benzene‐4‐carboxylic acid‐ and (CF) carboxyfluorescein‐labeled peptides. When the labeled peptide is intact, the fluorescence signal is weak thanks to the dark quenching property of DABCYL. A 10‐fold higher fluorescence signal is measured when the labeled peptide is degraded. By referring to a standard fluorescent curve according to the concentration of the hydrolyzed peptide, we have access to the internalized peptide quantity. Therefore, cell lysis after internalization permits to determine the total quantity of intracellular peptide. The molecular state of the internalized peptide (intact or degraded), depends on its location in cells (cytosol vs endo‐lysosomes), and can be blocked by boiling cells. This boiling step results indeed in denaturation and inhibition of the cellular enzymes. The advantage of this method is the possibility to quantify translocation at 37°C and to compare it to the 4°C condition, where all endocytosis processes are inhibited. We found that ranking of the translocation efficacy is DABCYL‐R6‐(εCF)K >> DABCYL‐R4‐(εCF)K ≥ CF‐R9.