The proteasome is essential for maintaining proteostasis and regulating a vast array of cellular functions, yet how proteasomal degradation is controlled within the complex cellular environment remains largely unclear. Proteasomal degradation is now understood not as a simple linear pathway, but as a multi-layered process involving intricate regulation by factors such as the p97 segregase, various shuttle molecules (e.g., RAD23A/B, UBQLNs), and the liquid-liquid phase separation (LLPS) of ubiquitinated substrates. Despite these advances, a fundamental challenge has persisted: the lack of a comprehensive method to quantitatively monitor substrate influx into the proteasome. To overcome this critical gap, we developed PSLip-MS, a novel technique that directly identifies and quantifies substrates captured within the proteasome's catalytic core. This talk will highlight the development of PSLip-MS and its application in elucidating the regulatory mechanisms of protein degradation under diverse conditions. By quantitatively mapping the proteasome substrate landscape, we are beginning to construct a systems-level model of how these multi-layered regulatory mechanisms converge to ensure cellular protein homeostasis. This research provides a powerful new technology and conceptual framework for achieving an integrated, systems-level understanding of proteasome degradation.