Identifying the peptides generated by the proteasome is essential for understanding its role in regulating critical cellular processes such as apoptosis, signaling, and immune responses. In this study, we introduce an enzymatic top-down mass spectrometry approach that allows real-time monitoring of proteasome-mediated cleavage, providing detailed insights into substrate degradation patterns. By examining proteasomes from different organs, we reveal organ-specific peptide production and degradation behaviors, offering a deeper understanding of how proteasome subtypes affect cellular function. This method is well-suited for investigating the influence of proteasome regulators, inhibitors and activators, on proteasome activity. Additionally, its versatility makes it applicable to other proteolytic complexes, paving the way for further research on proteostasis systems.