Abstract: Elucidating the structures of proteins and their variants is a fundamental prerequisite for understanding their biological functions and molecular regulatory mechanisms. Mass spectrometry (MS) has emerged as a powerful tool for large-scale, precise protein identification owing to its high sensitivity, high accuracy, and high-throughput capabilities. While bottom-up proteomics (BUP) typically involves enzymatic digestion that can lead to the loss of modification site information and low sequence coverage, top-down proteomics (TDP) analyzes intact proteins, effectively circumventing these limitations to provide comprehensive characterization of protein sequences and post-translational modifications. This paper systematically reviews the analytical workflow of TDP, covering key stages including sample preparation, ionization techniques, tandem mass spectrometry, and data processing. Addressing current technical bottlenecks, we highlight several breakthrough advancements: the integration of separation techniques such as reversed-phase liquid chromatography and capillary isoelectric focusing to effectively reduce sample complexity; the application of soft ionization techniques like electrospray ionization (ESI) to ensure protein structural integrity during mass spectrometry analysis; the combined use of multiple dissociation techniques to selectively cleave specific chemical bonds, enabling accurate localization of protein modification sites; and the continuous development of data preprocessing algorithms and protein identification tools, which has greatly improved the efficiency and accuracy of protein identification. Finally, future directions for the development of TDP technologies are discussed.