Metabolites are low molecular weight biomolecules and are important to biological systems, serving as building blocks of the cell membranes, proteome and genome. They play a crucial role as energy source, metabolic intermediates and molecules those have the ability of signalling. Their presence provide insights into biological functions and phenotypic responses to genetic or environmental changes. The field of metabolomics investigates these interactions and the roles of metabolites in research and clinical applications. Disruptions in metabolism are implicated in numerous genetic disorders, and many diseases are directly linked with adverse interactions between the genome, proteome and metabolome. Metabolomics has evolved with advancements in analytical instrumentation, employing techniques such as mass spectrometry (MS), nuclear magnetic resonance (NMR), and spectroscopy methods (Raman and Fourier Transform Infrared spectroscopy, FTIR). These techniques are used to profile metabolic changes and identify metabolic components. The surge in the need for analyzing low-level drugs in complex biological samples has highlighted the importance of sensitive and selective analytical methods for accurate quantification. This is critical for pharmacological studies, especially when the metabolite is pharmacologically active, toxic or its concentration in plasma equals or surpasses that of the parent drug. Thus, advancements in analytical techniques are a need of the hour for accurate identification and quantification of diverse metabolites for successful biomedical researches.