MHC class I molecules present peptides that are derived from intracellular proteins degraded by proteasomes. These peptides often require additional trimming by peptidases to fit into the peptide-binding grove of MHC class I. However, most peptides are rapidly recycled by the large heterogeneous pool of peptidases. Here, we describe a technique to quantify peptide degradation both in living cells and in cell lysates, using quenched peptides that contain a quencher and fluorophore. As degradation results in separation of the quencher and fluorophore, fluorescence will increase. This technique enables the examination of changes in peptide length and amino acid sequence on its half-life, and hence its chances to become presented by MHC class I.