The ability of some proteases to cleave polypeptide chains at restricted cleavage sites makes them important tools for the elucidation of the primary and even higher-order structure of proteins. This chapter provides an introduction into the methodology of enzymatic protein fragmentation. In addition, a series of complementary chemical methods are included which are indispensable for protein sequence analysis and peptide mapping. The specificity of most endopeptidases show a strong preference for particular amino acid residues or short sequence recognition sites. The chemical neighborhood of these residues, i.e. the character of their side chains, like hydrophobicity or size, may influence the rate of hydrolysis.