Many biologically active bacterial natural products contain highly modified deoxysugar residues that are often critical for the activity of the parent compounds. Most of these deoxysugars are secondary metabolites that are biosynthesized in the form of nucleotide diphosphate (NDP) sugars prior to their transfer to natural product aglycones by glycosyltransferases. Over the past decade, many biosynthetic pathways that lead to the formation of these unusual sugars have been unraveled, and the mechanisms of many key enzymatic transformations involved in these pathways have been elucidated. However, obtaining workable quantities of NDP-deoxysugars for in vitro studies is often a difficult task. This limitation has hindered an in-depth investigation of the substrate specificity of deoxysugar biosynthetic enzymes, many of which are promiscuous with respect to their NDP-sugar substrates and are, thus, potentially useful catalysts for natural product glycoengineering. Presented in this review are procedures for the enzymatic synthesis and purification of a variety of NDP-deoxysugars, including some early intermediates in NDP-deoxysugar biosynthetic pathways, and highly modified NDP-deoxysugars that are late intermediates in their respective biosynthetic pathways. The procedures described herein could be used as general guidelines for the development of specific protocols for the synthesis of other NDP-deoxysugars.