Penicillin-binding proteins (PBPs) are integral players in bacterial cell division, and their catalytic activities can be monitored with β-lactam-containing chemical probes. Compounds that target a single PBP could provide important information about the specific role(s) of each enzyme, making identification of such molecules important. We evaluated 22 commercially available β-lactams for inhibition of the PBPs in live Escherichia coli strain DC2. Whole cells were titrated with β-lactam antibiotics and subsequently incubated with a fluorescent penicillin derivative, Bocillin-FL (Boc-FL), to label uninhibited PBPs. Protein visualization was accomplished by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation and fluorescent scanning. The examined β-lactams exhibited diverse PBP selectivities, with amdinocillin (mecillinam) showing selectivity for PBP2, aztreonam, piperacillin, cefuroxime, cefotaxime, and ceftriaxone for PBP3, and amoxicillin and cephalexin for PBP4. The remaining β-lactams did not block any PBPs in the DC2 strain of E. coli or inhibited more than one PBP at all examined concentrations in this Gram-negative organism.