Advanced studies in molecular biology and biotechnology depends on the isolation and analysis of microbial molecules. This chapter presents an in-depth overview of key electrophoretic and chromatographic techniques used for biomolecular characterization. Electrophoresis, including PAGE, gradient gels, agarose gel electrophoresis, 2D electrophoresis, dielectrophoresis, and isoelectric focusing, allows for the resolution of nucleic acids and proteins based on size, charge, and isoelectric point. These techniques provide high-resolution separation essential for proteomic and genomic studies. Additionally, radioactive methods, including isotopic labeling, decay detection, sample preparation, and radioisotope counting, remain vital for tracing molecular pathways and quantifying biological activity. In addition to this, chromatographic methods make it possible to separate and analyze complicated biological mixtures efficiently, which is essential for metabolomics, medication development, and the identification of biomarkers. When combined, these techniques provide a thorough analytical comprehensive framework that facilitates accurate molecular research in cutting-edge biosciences.