Pancreatic cancer (PC), one of the most aggressive malignancies, is characterized by a dismal prognosis owing to its low early detection rates, rapid progression, frequent postoperative complications, and limited efficacy of conventional oncological therapies. The fact that most patients are diagnosed at advanced stages underscores the critical importance of early detection for the formulation of effective treatment strategies. Despite substantial research efforts, the medical community still lacks consistent and dependable biomarkers for the diagnosis, classification, and prognosis of PC, highlighting the urgent need for innovative and more efficient approaches to identify pancreatic abnormalities at early stages. For decades, mass spectrometry (MS)-based proteomics has been extensively applied in disease diagnostics, mechanistic investigations, and screening of potential drug targets. This review systematically synthesizes recent advancements in clinical proteomic techniques and applications, highlighting significant biomarker discoveries and signal transduction networks associated with PC. By integrating these findings, we provide novel insights into the molecular mechanisms underlying PC development and progression, which may facilitate the identification of new diagnostic biomarkers and therapeutic targets for this disease.© 2025. The Author(s).