Climate change and ecological invasions are altering temperature patterns and weather conditions, creating environments favorable for endemic pests, microbial pathogens, and soil-borne pathogens, particularly those with broad host ranges, which affect plant health and, consequently, environmentally sustainable crop production. Developing superior cultivars resistant to abiotic and biotic stresses through plant breeding faces numerous challenges. Plants have evolved primary defense mechanisms that produce secondary metabolites like terpenes, flavonoids, alkaloids, saponins, and glucosinolates upon pathogen attack, playing crucial roles in microbial defense infection. In recent years, pathologists have increasingly applied techniques in the omics field, such as genomic, proteomics, transcriptomics, volatilomics, spectranomics, and metabolomics to gain deeper insights into these interactions. The comprehensive analysis of small molecules or metabolites in cells, tissues, or organisms has become a valuable approach for investigating plant metabolites produced in response to pathogen attacks. In recent years, the emergence of next-generation sequencing, particularly whole genome sequencing (WGS), has transformed our understanding of plant-pathogen interactions, providing in-depth insights into the intricate biochemical networks that govern plant defense mechanisms. By integrating metabolomics with the NGS technologies which helps to sequence many samples within a short time will enable the researchers understand the specific metabolites involved in plant defense but also facilitate the identification of novel biomarkers and pathways that can be targeted for crop improvement. This chapter discusses the critical role of metabolomics in conjunction with next-generation approaches, highlighting its potential to transform plant pathology and crop protection research.