Now-a-days, top-down proteomics (TDP) is a booming approach for the analysis of intact proteins and it is attaining significant interest in the field of protein biology. The term has emerged as an alternative to the well-established, bottom-up strategies for analysis of peptide fragments derived from either enzymatically or chemically digestion of intact proteins. TDP is applied to mass spectrometric analysis of intact large biomolecules that are constituents of protein complexes and assemblies. This article delivers an overview of the methodologies in top-down mass spectrometry, mass spectrometry instrumentation and an extensive review of applications covering the venomics, biomedical research, protein biology including the analysis of protein post-translational modifications (PTMs), protein biophysics, and protein complexes. In addition, limitations of top-down proteomics, challenges and future directions of TDP are also discussed.