Pseudomonas aeruginosa is a Gram-negative, opportunistic pathogen that is a major cause of nosocomial infections in immunocompromised patients. Chronic P. aeruginosa infections are difficult to treat due to a limited sensitivity to antimicrobials resulting from an abundance of resistance mechanisms. For example, P. aeruginosa has the ability to transition from free roaming (planktonic) cells into biofilms, which provide protection against antimicrobials. P. aeruginosa also produces numerous antibiotic-inactivating enzymes such as β-lactamases, which target and break down β-lactam antibiotics. Through proteomic analysis of the P. aeruginosa laboratory strain PAO1, a number of uncharacterized proteins were identified that are predicted to be novel β-lactamases. This project focuses on characterizing three of the identified proteins: the biofilm dominant PA2915, the biofilm and planktonic variable PA0832, and the planktonic dominant PA0057. Targeted mutagenesis using allelic exchange was used to remove genes of interest in P. aeruginosa. The resulting knockouts were tested for susceptibility to β-lactam antibiotics as both biofilm and planktonic communities. In addition, the target proteins were biochemically characterized in vitro by detecting β-lactamase activity using the standard chromogenic β-lactam substrate, nitrocefin. These results show that PA2915 and PA0832 possess low level β-lactamase activity but do not provide any significant resistance against β-lactam antibiotics in P. aeruginosa. However, these proteins may play a role in biofilm biomass production in late-stage biofilm formation, as well as during carboxypenicillin stress. PA0057 activity both in vitro and in vivo was inconclusive. Overall, this work provides the foundation for future structure function studies of these putative β-lactamases.