Beta-lactam ring hydrolysis by beta-lactamases represents the primary resistance mechanism against beta-lactam antibiotics. Gram-negative bacteria employ two key protein translocation pathways across cytoplasmic membranes: the general secretory (Sec) and twin-arginine translocation (Tat) pathways. While most beta-lactamases utilize the Sec pathway, this study identifies a significant cluster of Tat-dependent beta-lactamases. A bioinformatic approach is presented to predict Tat-dependence, followed by biochemical validation and characterization using isogenic Y. enterocolitica and E. coli models.