Flavonoids are polyphenolic secondary metabolites with various in vivo and in vitro structure-related benefits that are ubiquitously found in plants. In addition to their role in many physiological activities, flavonoids play major roles as phytoalexins and phytoanticipins in protecting plants from potent pathogens. Since the first discovery of such defense roles, their routes of biosynthesis induced upon pathogen infection and mechanisms of action have attracted the attention of researchers in various fields. The ultimate goal of the study of plant-pathogen interactions is to develop biotechnological applications that enhance crop production in an environmentally friendly manner, with the outcome of engineering highly pathogen-resistant genotypes. Steady progress has been made previously in this regard. Inoculation of potential pathogens in plant tissues, followed by the systematic analysis of various targeted biochemical changes, including investigations into flavonoid variations, has become an appealing methodological approach. In this chapter, the role of flavonoids as defense mechanisms in plant-pathogen interactions is discussed with respect to the latest findings. In addition, we emphasize research trends observed in this regard. In light of the most recent studies, including those conducted by some of the authors of this chapter, key findings in flavonoid variations upon pathogen infection are highlighted comprehensively, focusing on fungal inoculation and liquid chromatography-tandem mass spectrometry analysis for reasons described in later sections.