Transpeptidation as the last step in peptidoglycan synthesis has been a successful antibacterial target for many decades, whereas inhibitors of the preceding transglycosylation did not lead to any commercialized drugs although both reactions are essential for cell survival. Since the substrate, Lipid II, has become more easily available, a lot of research towards synthesis of new inhibitors of transglycosylation has been done. Several assay types have been elaborated to evaluate them. The main problem in the development is the lack of UV-chromophores in the substrate leading to the need for radioactive and fluorescence labeling. Also, high-throughput screens that allow screening of large libraries of compounds have been conceived. This review gives an overview of the different assay types and highlights some cleverly engineered screens.