The advancement in the field of microbial biotechnology depends upon the precise separation and analysis of biomolecules, which can be achieved through techniques such as electrophoresis, chromatography, and radioactive methods. Use of these techniques for analysis of biomolecules depends mainly upon experimental method, cost-effectiveness, and analysis time. Electrophoresis has shown potential for high-resolution separation of nucleic acid; however, more development is required to reduce the requirement of highly concentrated samples, particularly for larger nucleic acids. Among chromatographic methods, liquid chromatography (LC) is the most widely used separation method; it is crucial for the separation of contaminants like degradation products. Integration of mass spectroscopy with LC has enhanced the sensitivity and chromatographic resolution, enabling compound identification. These methods also help with environmental monitoring, food safety, and impurity profiling of various pharmaceutical products, which might cause a detrimental effect on human health. Further, radio-labeling has been done intensively for tracking and analysis of biomolecules, which require a multistep method for the incorporation of radioactive isotopes. These advanced methodologies are now vital for strain identification, metabolite quantification, and drug discovery. As the field of microbial biotechnology expands, these techniques remain at the leading methods for driving discovery and innovation across diverse scientific and industrial domains.