The past decade has seen increased efforts to design biosensors for studying cellular mechanisms, bioengineering synthetic modules, and constructing diagnostic devices1. Protein-based biosensors that are chimeric protein switches are promising candidates for the aforementioned applications. For these purposes, a generalizable protein biosensor architecture based on engineered PQQ-glucose dehydrogenase (GDH) and calmodulin (CaM) chimera was developed by Guo et al2,3. GDH-CaM chimeric protein biosensor can be modified for the detection of many different analytes, which can then be translated into electrochemical Point-of-Care tests similar to ubiquitous glucometers. However, the current response time of GDH-CaM biosensor is in the order of minutes, while the ideal diagnostic device should return results in seconds. Hence in this study, we aimed to optimize reaction rate, dynamic range, and response time of GDH-CaM biosensor. For this purpose, we developed all in vitro expression-based platform for activity screening of domain insertion libraries that allows biochemical analysis in medium throughput (between 1000-2000 clones). About 23% of the library corresponding to 50 chimeras were found to be activated by calmodulin-binding peptides. When we statistically examined the active chimeras' performance parameters, it was observed that their dynamic range and response times are anticorrelated, indicating a thermodynamic trade-off. Moreover, one candidate resulted in 2-times higher reaction rate and 4-times faster response time than the previously best variant4. This work lays ground for optimization of response parameters intrinsic to many other protein-based biosensors.