In microbial biotechnology, the separation, detection, and analysis of biological molecules such as DNA, proteins, and metabolites are fundamental for understanding and engineering microbial systems. This chapter provides a comprehensive overview of biomolecular separation and analysis methods such as electrophoresis and chromatography in resolving complex biomolecular mixtures. Here, we discuss principles and applications of agarose-gel electrophoresis, polyacrylamide-gel electrophoresis (PAGE), gradient gel electrophoresis, and two-dimensional electrophoresis (2D-PAGE). This chapter also covers radioactive methods, such as the use of radioactive isotopes, sample preparation, and counting, which are crucial for sensitive detection and quantification in microbial studies. These analytical techniques are essential for studying various biological processes such as gene expression and protein profiling and for tracing biochemical pathways, labeling molecules, and quantifying biomolecular interactions. By integrating classical and advanced biomolecular approaches, this chapter provides a foundational and practical understanding of analytical techniques that are indispensable in modern microbial biotechnology research and applications.