Abstract Bacillus thuringiensis (Bt) Cry protein-based insect control has proven to be effective in reducing the use of chemical insecticides and in increasing crop yields. However, Bt crops increase selection pressure for resistant insects and accelerate their succession. The discovery and application of different Bt toxins that have no cross-resistance with known toxins has been proposed as a strategy for the management of resistant insects. Additionally, the discovery of novel Bt toxins with a new insecticidal spectrum will control insect succession. In fact, the discovery of new Bt toxins is one of the most important areas in Bt research, and the most advanced molecular biology methods available have been applied to this task. In this chapter, we summarize the published methods for Bt toxin discovery.