Abstract: Elucidating the structures of proteins and proteoforms is a fundamental prerequisite for understanding their biological functions and molecular regulatory mechanisms. Owing to its high sensitivity, accuracy, and throughput, mass spectrometry enables large-scale and precise identification of proteins. Mass spectrometry-based top-down proteomics (TDP) directly analyzes intact proteins, circumventing the loss of connectivity among peptide-derived information caused by enzymatic digestion in conventional bottom-up proteomics (BUP). Consequently, TDP preserves the combinatorial relationships among various structural variations and modifications at the proteoform level, compensating for the limitations of BUP in comprehensive proteoform characterization. This review systematically summarizes the complete analytical workflow of TDP, covering major steps such as sample preparation, ionization techniques, tandem mass spectrometry, and data processing, with particular emphasis on tandem mass spectrometry dissociation techniques. Different dissociation/ion activation methods, including ECD/ETD, AI-ETD, and UVPD, possess distinct fragmentation mechanisms and produce different types of fragment ions. Their combined application can substantially improve protein sequence coverage, enhance the confidence of post-translational modifications localization, and deepen the characterization of complex proteoform. Building upon these technological advances, TDP has demonstrated unique advantages in identifying disease-associated proteoform, elucidating pathological mechanisms, and discovering novel biofluid diagnostic biomarkers. This review also highlights the applications of TDP in cancer biomarker discovery, neurodegenerative diseases, and clinical diagnostics.