Background <p>Advancements in the research of intracellular metabolome have the potential to affect our understanding of biological processes. The applications and findings of intracellular metabolome analysis are useful in understanding cellular pathways, microbial interactions, and the detection of secreted metabolites and their functions.</p> Aim of review <p>This work focuses on the analysis of intracellular metabolomes in microorganisms. The techniques used for analyzing the intracellular metabolomes including metabolomics approaches such as mass spectrometry, nuclear magnetic resonance, liquid chromatography, and gas chromatography are discussed.</p> Key scientific concepts of review <p>Challenges such as sample preparation, data analysis, metabolite extraction, sample storage and collection, and processing techniques were investigated, as they can highlight emerging technologies and advancements in metabolome analysis, future applications in drug discovery, personalized medicine, systems biology, and the limitations and challenges in studying the metabolome of microorganisms.</p> Graphical abstract <p></p>