The expression of β-lactamases, particularly carbapenemases and extended-spectrum β-lactamases, is a direct threat to the efficacy of β-lactams, which represent over half of clinically used antibiotics. Currently available β-lactamase inhibitors are mechanism-based inhibitors of serine β-lactamases, and ineffective against metallo-β-lactamases. Concerningly, serine β-lactamases such as the carbapenemase KPC-2 from Klebsiella pneumoniae, and the AmpC cephalosporinase are resistant to most of these inhibitors. An improved understanding of the general assembly of this important class of enzymes has the potential to reveal additional drug targets to mitigate this significant mode of resistance. Some indirect evidence suggests that the periplasmic chaperone Spy and the DD-carboxypeptidase DacD may play a role in the periplasmic stability of β-lactamases. This study explores the influence of periplasmic chaperones on the folding of KPC-2 and AmpC, which were over-expressed in a series of E.coli knockouts lacking selected periplasmic chaperones. Strains were screened for reduced β-lactamase activity based on their ability to hydrolyze the chromogenic β-lactam substrate nitrocefin in order to identify chaperones that play a role in β-lactamase folding. Selected candidates were then subjected to a pulse-chase assay over a short time course to monitor changes in the periplasmic assembly products of KPC-2 and AmpC. Further investigations should unearth greater insight into the biogenesis of β-lactamases.