During the past decade metabolomics has emerged as one of the fastest developing branches of “-omics” technologies. Metabolomics involves documentation, identification, and quantification of metabolites through modern analytical platforms in various biological systems. Advanced analytical tools, such as gas chromatography–mass spectrometry (GC/MS), liquid chromatography–mass spectroscopy (LC/MS), and non-destructive nuclear magnetic resonance (NMR) spectroscopy, have facilitated metabolite profiling of complex biological matrices. Metabolomics, along with transcriptomics, has an influential role in discovering connections between genetic regulation, metabolite phenotyping and biomarkers identification. Comprehensive metabolite profiling allows integration of the summarized data towards manipulation of biosynthetic pathways, determination of nutritional quality markers, improvement in crop yield, selection of desired metabolites/genes, and their heritability in modern breeding. Along with that, metabolomics is invaluable in predicting the biological activity of medicinal plants, assisting the bioactivity-guided fractionation process and bioactive leads discovery, as well as serving as a tool for quality control and authentication of commercial plant-derived natural products. Metabolomic analysis of human biofluids is implemented in clinical practice to discriminate between physiological and pathological state in humans, to aid early disease biomarker discovery and predict individual response to drug therapy. Thus, metabolomics could be utilized to preserve human health by improving the nutritional quality of crops and accelerating plant-derived bioactive leads discovery through disease diagnostics, or through increasing the therapeutic efficacy of drugs via more personalized approach. Here, we attempt to explore the potential value of metabolite profiling comprising the above-mentioned applications of metabolomics in crop improvement, medicinal plants utilization, and, in the prognosis, diagnosis and management of complex diseases.