The ability to resolve effectively peptides obtained by chemical or proteolytic cleavage is important in the structural investigation of proteins, whether it be minute amounts of protein isolated from a biological source or protein produced recombinantly for pharmaceutical use. Capillary electrophoresis (CE) can be used rapidly to resolve peptides electrophoretically in free solution with a selectivity that differs from that of other separation approaches. In this article, we provide a description of the instrumentation for CE and the fundamental principles driving peptide separation at low and high pH. Guidelines for method optimization of peptide injection and separation by CE are given along with a number of examples from the literature demonstrating the utility of CE for interrogating protein structure via CE‐based peptide and glycopeptide analysis.