In this chapter, we provide a brief overview of the current status, challenges and scope for charge density analysis in proteins. The approach, basic requirements, available tools for performing protein charge density analysis are discussed here. A brief review of reported charge density studies on six protein systems ranging from 4.7 to 55 kDa and with resolutions ranging from 0.78 to 0.48 Å, and the key outcomes, which were inaccessible from the routine structural studies, are highlighted. The use of neutron diffraction data and its joint refinement with X-ray diffraction data for quantifying interactions involving water molecules themselves and with the protein residues is also discussed for a lone reported protein complex. Further, we perform a topological analysis of main-chain N-HO=C hydrogen bonds both in α-helices and β-sheets of a high-resolution (0.66 Å) protein structure of hAR based on electron densities from the ELMAM2 database. The quantitative characterizations reveal that the N-HO=C type hydrogen bonds in proteins follow the same trend as those in small molecules. Furthermore, both the electrostatic and dissociation energies of H-bonds are estimated based on the electron densities and electrostatic potentials. The electrostatic energies are comparable with the experimental values. The energies were also found to correlate well with the topological properties of N-HO=C hydrogen bonds. © The Royal Society of Chemistry 2019.