PenP is a fluorescent biosensor of lactam antibiotics (LA). It is structurally derived from the mutant lactamase TEM-1 comprising the substitution E166C, where fluorescein is covalently linked to cysteine. The presence of LA in the medium produces a change in the intrinsic fluorescence level of the biosensor, and the integral of the fluorescence level over time correlates directly with the LA concentration. Previously, we have successfully used PenP to determine the concentration of lactam antibiotics in clinical samples. The use of lactamase inhibitors (LI) is a common strategy to enhance the effect of LA due to the inhibition of an important resistance mechanism of pathogenic microorganisms. Structurally, LI and LA share the common element of recognition of lactamases (the lactam ring), but they differ in the reversibility of the mechanism of interaction with said enzyme. Because the biological recognition domain of PenP is derived from a lactamase, LI is expected to interfere with the PenP detection capabilities. Surprisingly, this work provides evidence that the effect of LI is marginal in the determination of LA concentration mediated by PenP.