Target identification is a crucial step in drug discovery, enabling the development of more effective and safer therapeutic agents. This study focuses on the development and validation of a novel bacterial three-hybrid (B3H) system designed to identify protein targets of small molecules. The system employs SNAP-tag technology to detect interactions between small molecules and their target proteins in Escherichia coli. The use of the Gateway recombination technique streamlined the cloning of fusion proteins and enhanced the system's flexibility. By combining the specificity of affinity methods with the versatility of genetic approaches, the B3H system provides a rapid and cost-effective way to study these interactions. Several tests confirmed the system's effectiveness by evaluating interactions with known targets such as Trypanosoma brucei adenosine kinase, avidin, and dihydrofolate reductase. This work paves the way for future applications in drug target discovery, particularly for bacterial systems.