Biofilm is a complex, structured community of bacteria that adhere to surfaces and are embedded in a self-produced matrix of Exopolysaccharide (EPS) and other cell wall-associated components. Biofilm formation provides increased resistance to temperature, gastric pH, and mechanical forces compared to their planktonic counterparts, thereby facilitating microbial communication, growth, and survival. The biofilm-forming phenotype of probiotic bacteria enhances bacterial adhesion to target sites by improving cell surface properties, thereby creating colonization resistance against pathogenic bacteria. Hence, detecting bacterial biofilm formation ability is crucial, as biofilms play a significant role in bacterial survival, virulence, and resistance to various physical and chemical treatments. This chapter describes various promising in vitro preliminary and microscopic test protocols to identify the biofilm formation ability of bacteria.