Inhibitors of transglycosylases may serve as potent antibiotics that are less prone to resistance development in bacterial pathogens. To facilitate the search of such compounds, a transglycosylase (TGase) domain of the membrane integral multidomain Streptococcus pneumoniae PBP1b was cloned and expressed. This TGase domain was characterized by a substrate-dependent fluorescence coupled enzyme assay and an inhibitor-tethered surface plasmon resonance binding assay. Both assays show that the catalytic efficiency of the domain is comparable to that of the monofunctional transglycosylases, and it is fully active in the absence of other domains. The isolation of the active TGase domain makes it possible to screen for potential antibiotics targeting transglycosylases.