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Western blotting (WB), also known as immunoblotting, is a well-known molecular biology method that biologists often use to investigate many features of the protein, ranging from basic protein analysis to disease detection. WB is simple, unique, rapid, widely used routine tool with easy interpretation and definite results. It is being used in various fields of science, research and development, diagnostic labs and hospitals. The principle of WB is to accomplish the separation of proteins based on molecular weight and charge. This review addresses in detail the individual steps involved in the WB technique, its troubleshooting, internal loading controls, total protein staining and its diverse applications in scientific research and clinical settings, along with its future perspectives.