D-Ala-Lys-AMCA hydrochloride is a known proton-coupled oligopeptide transporter 1 (PEPT1) substrate that emits blue fluorescence. D-Ala-Lys-AMCA hydrochloride may be transported into liver cancer cells and Caco-2 cells based on fluorescence analysis. D-Ala-Lys-AMCA hydrochloride can be used for characterizing PEPT1-specific substrates or inhibitors.