The application of fungicides is a measure complementary to host genetic resistance to control the occurrence and severity of rust diseases that has been estimated to cost over $17.25 per acre annually in wheat fields on the Canadian Prairies. The most often used fungicides include the class of demethylation inhibitors (DMIs), acting on fungal sterol biosynthesis, and the class of strobilurins (quinone outside [mitochondrial respiration] inhibitor [QoI]) acting on fungal mitochondrial respiration. Fungicides are designed to target fungal pathogens but also have been reported to trigger some effects on the host plants. Therefore, an improved diagnostic protocol is developed in this chapter for evaluating the effects and efficacy of commercial fungicides: DMI and QoI on in vitro germination of rust fungus urediniospores and rust disease development of infected, detached wheat leaves as well as whole plants. The purpose is to optimize fungicide application to better control rust fungus diseases of wheat without impacting crop growth, and while mitigating fungicide applications to minimize environmental and financial costs associated with fungicide overapplication.